Argonaute HITS-CLIP Reveals Global miRNA-mRNA Networks in Erythropoiesis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 446-446 ◽  
Author(s):  
Vikram R Paralkar ◽  
Jing Luan ◽  
Srijani Sridhar ◽  
Anastassios Vourekas ◽  
Zissimos Mourelatos ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mrna Stability ◽  
Mirna Target ◽  
Fetal Liver ◽  
Erythroid Cells ◽  
Protein Coding ◽  
Target Mrna ◽  
Target Mrnas ◽  
Primary Mouse ◽  
In Erythroid Cells

Abstract MicroRNAs (miRNAs) inhibit gene expression by recruiting the RNA-induced silencing complex (RISC) to specific sets of target mRNAs. However, it has been challenging to define precisely the miRNA-target mRNA interactions that occur within cells. One approach to rigorously characterizing these networks in cells of interest is to sequence mRNA fragments bound to the RISC component Argonaute using a method termed High-Throughput Sequencing of RNA isolated by Crosslinking Immunoprecipitation (HITS-CLIP). We used this approach to define miRNA-target mRNA interactions during erythropoiesis, a developmental process known to be regulated by miRNAs and dramatically impaired by loss of Argonaute function. We performed Argonaute HITS-CLIP on primary mouse fetal liver erythroblasts in 3 biological replicate experiments and used Bowtie tools to map the sequenced RNA fragments to the mouse genome. These sequences represent mature miRNAs and their bound RNA segments in erythroid cells. Remarkably, miR-451 accounted for 70% of the Ago-bound erythroid miRNA burden, with miR-142, miR-144, miR-21, miR-374 and miR-30 accounting for an additional 15%. The frequency of Ago-binding correlated well with total cellular miRNA abundance. To analyze miRNA-target interactions, we used Piranha tools to identify peaks of sequenced reads, focusing on those that map to protein coding mRNAs. We identified 3,670 peaks across the entire genome, most of which mapped to mRNA 3’ untranslated regions (UTRs) (45%) or protein coding regions (38%). The remaining peaks mapped to intergenic regions, introns, long noncoding RNA genes and pseudogenes. To identify miRNA-mRNA associations, we searched for base pair complementarity between HITS-CLIP peaks mapping to mRNAs and seed sequence matches to the top 20 miRNAs in erythroid cells, which account for 92% of total miRNAs. We matched 36% of all peaks to canonical seed sequences and 34% to non-canonical seed sequences of specific miRNAs. We compared miR451-target mRNA matches predicted by HITS-CLIP to mRNA expression datasets generated from wild-type (WT) and miR-144/451 knockout (KO) mouse fetal liver erythroblasts. mRNAs with canonical miR-451 peaks in 3’ UTRs were significantly upregulated in KO erythroblasts as compared to WT erythroblasts (p<0.0001), indicating that our HITS-CLIP study identifies true erythroid targets of miR-451. These include previously identified targets of miR-451 such as Cab39, Ywhaz and Vapa, further validating our study. We also identified previously unknown targets such as Copa and Reep5, which, along with Vapa, regulate vesicle trafficking in other cell systems, and Matr3, Patl1 and Ythdf2, which affect global mRNA stability. Study of these genes may provide further insight into the functions of miR-451 in erythropoiesis. In contrast, mRNAs with predicted miR-451 peaks within coding exons showed no change in expression. This indicates that not all Ago HITS-CLIP peaks reflect regulation of mRNA stability, and that the presence of 3’ UTR Ago peaks with canonical miRNA seed sequences predicts better this mode of post transcriptional control of gene expression. Analysis and comparison of mRNA translational regulation by 3’UTR and coding region-bound miRNAs is in progress. Overall, our results provide a comprehensive map of Ago-bound miRNAs and their biologically relevant target mRNAs in erythroid cells. This map will serve as a basis to better understand miRNA regulation of erythropoiesis. Disclosures No relevant conflicts of interest to declare.

scholarly journals Developmental Expression of Mouse Erythrocyte Protein 4.2 mRNA: Evidence for Specific Expression in Erythroid Cells

Blood ◽  
1998 ◽  
Vol 91 (2) ◽  
pp. 695-705 ◽  
Author(s):  
Lingyun Zhu ◽  
Samir B. Kahwash ◽  
Long-Sheng Chang
Keyword(s):  
Mrna Expression ◽  
Fetal Liver ◽  
Late Gestation ◽  
Developmental Expression ◽  
Erythroid Cells ◽  
Mouse Development ◽  
Specific Expression ◽  
Protein 4.2 ◽  
Specific Regulation ◽  
In Erythroid Cells

Abstract Erythrocyte protein 4.2 (P4.2) is an important component of the erythrocyte membrane skeletal network with an undefined biologic function. Presently, very little is known about the expression of the P4.2 gene during mouse embryonic development and in adult animals. By using the Northern blot and in situ hybridization techniques, we have examined the spatial and temporal expression of the P4.2 gene during mouse development. We show that expression of the mouse P4.2 gene is temporally regulated during embryogenesis and that the P4.2 mRNA expression pattern coincides with the timing of erythropoietic activity in hematopoietic organs. P4.2 transcripts are first detected in embryos on day 7.5 of gestation and are localized exclusively in primitive erythroid cells of yolk sac origin. These erythroid cells remain to be the only source for P4.2 expression until the switch of the hematopoietic producing site to fetal liver. In mid- and late-gestation periods, P4.2 mRNA expression is restricted to the erythroid cells in fetal liver and to circulating erythrocytes. Around and after birth, the site for P4.2 expression is switched from liver to spleen and bone marrow, and P4.2 transcripts are only detected in cells of the erythroid lineage. These results provide the evidence for specific P4.2 expression in erythroid cells. In addition, the timing and pattern of expression of the P4.2 gene suggest the specific regulation of the P4.2 gene.

scholarly journals Developmental- and differentiation-specific patterns of human γ- and β-globin promoter DNA methylation

Blood ◽  
2007 ◽  
Vol 110 (4) ◽  
pp. 1343-1352 ◽  
Author(s):  
Rodwell Mabaera ◽  
Christine A. Richardson ◽  
Kristin Johnson ◽  
Mei Hsu ◽  
Steven Fiering ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Promoter Methylation ◽  
Fetal Liver ◽  
Globin Gene ◽  
Erythroid Cells ◽  
Promoter Dna Methylation ◽  
Globin Promoter ◽  
Promoter Dna

AbstractThe mechanisms underlying the human fetal-to-adult β-globin gene switch remain to be determined. While there is substantial experimental evidence to suggest that promoter DNA methylation is involved in this process, most data come from studies in nonhuman systems. We have evaluated human γ- and β-globin promoter methylation in primary human fetal liver (FL) and adult bone marrow (ABM) erythroid cells. Our results show that, in general, promoter methylation and gene expression are inversely related. However, CpGs at −162 of the γ promoter and −126 of the β promoter are hypomethylated in ABM and FL, respectively. We also studied γ-globin promoter methylation during in vitro differentiation of erythroid cells. The γ promoters are initially hypermethylated in CD34+ cells. The upstream γ promoter CpGs become hypomethylated during the preerythroid phase of differentiation and are then remethylated later, during erythropoiesis. The period of promoter hypomethylation correlates with transient γ-globin gene expression and may explain the previously observed fetal hemoglobin production that occurs during early adult erythropoiesis. These results provide the first comprehensive survey of developmental changes in human γ- and β-globin promoter methylation and support the hypothesis that promoter methylation plays a role in human β-globin locus gene switching.

scholarly journals Substitution of the Human β-Spectrin Promoter for the Human Aγ-Globin Promoter Prevents Silencing of a Linked Human β-Globin Gene in Transgenic Mice

1998 ◽  
Vol 18 (11) ◽  
pp. 6634-6640 ◽  
Author(s):  
Denise E. Sabatino ◽  
Amanda P. Cline ◽  
Patrick G. Gallagher ◽  
Lisa J. Garrett ◽  
George Stamatoyannopoulos ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Fetal Liver ◽  
Globin Gene ◽  
Gene Promoter ◽  
Yolk Sac ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Globin Mrna ◽  
Globin Promoter ◽  
In Erythroid Cells

ABSTRACT During development, changes occur in both the sites of erythropoiesis and the globin genes expressed at each developmental stage. Previous work has shown that high-level expression of human β-like globin genes in transgenic mice requires the presence of the locus control region (LCR). Models of hemoglobin switching propose that the LCR and/or stage-specific elements interact with globin gene sequences to activate specific genes in erythroid cells. To test these models, we generated transgenic mice which contain the human Aγ-globin gene linked to a 576-bp fragment containing the human β-spectrin promoter. In these mice, the β-spectrin Aγ-globin (βsp/Aγ) transgene was expressed at high levels in erythroid cells throughout development. Transgenic mice containing a 40-kb cosmid construct with the micro-LCR, βsp/Aγ-, ψβ-, δ-, and β-globin genes showed no developmental switching and expressed both human γ- and β-globin mRNAs in erythroid cells throughout development. Mice containing control cosmids with the Aγ-globin gene promoter showed developmental switching and expressed Aγ-globin mRNA in yolk sac and fetal liver erythroid cells and β-globin mRNA in fetal liver and adult erythroid cells. Our results suggest that replacement of the γ-globin promoter with the β-spectrin promoter allows the expression of the β-globin gene. We conclude that the γ-globin promoter is necessary and sufficient to suppress the expression of the β-globin gene in yolk sac erythroid cells.

Gene Expression and Biological Significance of Hexokinase in Erythroid Cells

2002 ◽  
Vol 108 (4) ◽  
pp. 204-209 ◽  
Author(s):  
Koko Murakami ◽  
Hitoshi Kanno ◽  
Jakica Tancabelic ◽  
Hisaichi Fujii
Keyword(s):  
Gene Expression ◽  
Biological Significance ◽  
Erythroid Cells ◽  
In Erythroid Cells

Regulation Of Erythro-Megakaryocytic Lineage Bifurcation By The Gfi1b Gene Target Rgs18

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1191-1191
Author(s):  
Ananya Sengupta ◽  
Ghanshyam Upadhyay ◽  
Asif Chowdhury ◽  
Shireen Saleque
Keyword(s):  
Conflicts Of Interest ◽  
Integration Site ◽  
Fetal Liver ◽  
Cell Types ◽  
Mapk Signaling ◽  
Erythroid Cells ◽  
Erythroid Progenitor ◽  
Gene Target ◽  
Over Expression ◽  
In Erythroid Cells

Abstract The molecular basis for the divergence of the erythroid (red blood cell) and megakaryocyte (platelet) lineages from a common bipotent MEP (megakaryocyte-erythroid progenitor) remains undefined. We now demonstrate that Rgs18 (regulator of G protein signaling 18), a GAP (GTPase activating protein) factor and a transcriptional gene target of the Gfi1b transcriptional repressor complex, likely arbitrates this critical lineage decision downstream of Gfi1b. Rgs18 was identified in a chromatin immunoprecipitation (ChIP on chip) screen for Gfi1b/LSD1/Rcor1 targets in erythroid cells. Accordingly, Rgs18 expression was found to be up-regulated in LSD1 inhibited, and Gfi1b deficient erythroid cells confirming repression of this gene by Gfi1b and its co-factors in this lineage. In contrast, Rgs18 expression was comparable in megakaryocytic cells derived from wild type and gfi1b-/-hematopoietic progenitors indicating Gfi1b independent expression of Rgs18 in these cells. Manipulation of Rgs18 expression produced opposite effects in the erythroid and megakaryocytic lineages. Rgs18 inhibition retarded megakaryocytic differentiation while its ectopic over-expression promoted differentiation at the expense of proliferation. The reverse was observed in erythroid cells where Rgs18 inhibition produced an enhancement of differentiation while over-expression impaired erythropoiesis. Since Rgs signaling regulates the activity of downstream MAPK pathways we determined the status of these pathways in Rgs18 manipulated cells. Inhibition of Rgs18 stimulated ERK phosphorylation in megakaryocytes but diminished it in erythroid cells. In contrast, Rgs18 inhibition in erythroid cells elevated p38MAPK protein and phosphorylation levels. The opposite effects of Rgs18 manipulation on MAPK signaling in erythroid versus megakaryocytic cells while intriguing are consistent with the changes in differentiation and proliferation observed in each lineage, respectively. Although Rgs18 manipulation produced opposite effects in erythroid and megakaryocytic cells, the level and activity of this factor correlated similarly with those of the mutually antagonistic transcription factors Fli1 (Friend leukemia integration [site] 1) and KLF1/EKLF (Kruppel like factor1) in both cell types. In both lineages, Rgs18 protein levels correlated directly with Fli1, and inversely with KLF1, message levels. Since Fli1 promotes megakaryocytic, and KLF1 erythroid, development; these results demonstrate that Rgs18 promotes the emergence of megakaryocytic cells from bipotent MEPs by modulating MAPK signaling and altering Fli1/KLF1 stoichiometries. Although it is unclear why Gfi1b mediated repression of Rgs18 is erythroid specific even though the former is expressed in both lineages, these results demonstrate that repression of Rgs18 by Gfi1b in fetal liver MEPs limits megakaryopoiesis and augments erythropoiesis. However following megakaryocytic commitment, robust Gfi1b independent expression of Rgs18 drives differentiation of this lineage while continued repression of Rgs18 by Gfi1b in erythroid cells ensures their proper maturation. Disclosures: No relevant conflicts of interest to declare.

mRNA-protein complexes controlling gene expression in erythroid cells

2005 ◽  
Vol 2005 (Fall) ◽  
Author(s):  
Dirk Ostareck ◽  
Christiane Harnisch ◽  
Antje Ostareck-Lederer
Keyword(s):  
Gene Expression ◽  
Protein Complexes ◽  
Erythroid Cells ◽  
In Erythroid Cells

scholarly journals Antagonistic Regulation of β-Globin Gene Expression by Helix-Loop-Helix Proteins USF and TFII-I

2006 ◽  
Vol 26 (18) ◽  
pp. 6832-6843 ◽  
Author(s):  
Valerie J. Crusselle-Davis ◽  
Karen F. Vieira ◽  
Zhuo Zhou ◽  
Archana Anantharaman ◽  
Jörg Bungert
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Control Region ◽  
Adult Stage ◽  
Globin Gene ◽  
Locus Control Region ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Globin Gene Expression ◽  
In Erythroid Cells

ABSTRACT The human β-globin genes are expressed in a developmental stage-specific manner in erythroid cells. Gene-proximal cis-regulatory DNA elements and interacting proteins restrict the expression of the genes to the embryonic, fetal, or adult stage of erythropoiesis. In addition, the relative order of the genes with respect to the locus control region contributes to the temporal regulation of the genes. We have previously shown that transcription factors TFII-I and USF interact with the β-globin promoter in erythroid cells. Herein we demonstrate that reducing the activity of USF decreased β-globin gene expression, while diminishing TFII-I activity increased β-globin gene expression in erythroid cell lines. Furthermore, a reduction of USF activity resulted in a significant decrease in acetylated H3, RNA polymerase II, and cofactor recruitment to the locus control region and to the adult β-globin gene. The data suggest that TFII-I and USF regulate chromatin structure accessibility and recruitment of transcription complexes in the β-globin gene locus and play important roles in restricting β-globin gene expression to the adult stage of erythropoiesis.

scholarly journals ETO-2 Associates with SCL in Erythroid Cells and Megakaryocytes and Provides Repressor Functions in Erythropoiesis

2005 ◽  
Vol 25 (23) ◽  
pp. 10235-10250 ◽  
Author(s):  
Anna H. Schuh ◽  
Alex J. Tipping ◽  
Allison J. Clark ◽  
Isla Hamlett ◽  
Boris Guyot ◽  
...  
Keyword(s):  
Gene Expression ◽  
Target Genes ◽  
Protein Complexes ◽  
Fetal Liver ◽  
Regulation Of Gene Expression ◽  
Erythroid Differentiation ◽  
Cell Types ◽  
Erythroid Cells ◽  
Proteomic Approach ◽  
Endogenous Proteins

ABSTRACT Lineage specification and cellular maturation require coordinated regulation of gene expression programs. In large part, this is dependent on the activator and repressor functions of protein complexes associated with tissue-specific transcriptional regulators. In this study, we have used a proteomic approach to characterize multiprotein complexes containing the key hematopoietic regulator SCL in erythroid and megakaryocytic cell lines. One of the novel SCL-interacting proteins identified in both cell types is the transcriptional corepressor ETO-2. Interaction between endogenous proteins was confirmed in primary cells. We then showed that SCL complexes are shared but also significantly differ in the two cell types. Importantly, SCL/ETO-2 interacts with another corepressor, Gfi-1b, in red cells but not megakaryocytes. The SCL/ETO-2/Gfi-1b association is lost during erythroid differentiation of primary fetal liver cells. Genetic studies of erythroid cells show that ETO-2 exerts a repressor effect on SCL target genes. We suggest that, through its association with SCL, ETO-2 represses gene expression in the early stages of erythroid differentiation and that alleviation/modulation of the repressive state is then required for expression of genes necessary for terminal erythroid maturation to proceed.

scholarly journals The Condensin II Complex Subunit NCAPH2 Is Essential for Erythropoiesis and Regulates Erythroid Gene Expression

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 286-286
Author(s):  
Zachary C. Murphy ◽  
Maeve Wells ◽  
Kristin Murphy ◽  
Michael Getman ◽  
Laurie A. Steiner
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Conflicts Of Interest ◽  
Chromatin Condensation ◽  
Differentially Expressed ◽  
Erythroid Cells ◽  
Dramatic Decline ◽  
Upregulated Genes ◽  
Mutant Cells ◽  
In Erythroid Cells

Abstract Erythropoiesis requires dramatic changes in gene expression in a cell that is rapidly proliferating and undergoing progressive nuclear condensation in anticipation of enucleation. Disruption of this process is associated with myelodysplastic syndromes and congenital anemias. Our lab has demonstrated that Setd8, the sole histone methyltransferase that can generate H4K20me1, plays an essential role in this process (Malik 2019). H4K20me1 accumulates during terminal erythroid maturation (Murphy Blood 2021) and can regulate chromatin structure and gene expression through interaction with multiple partners, including the Condensin II Complex. The Condensin II complex is a ring-like structure composed of two conserved SMC components (SMC2 and SMC4), two HEAT subunits (NCAPG2 and NCAPD3), and a kleisin subunit NCAPH2. The Condensin II complex plays an important role in chromatin condensation during mitosis, and establishing higher-order chromatin interactions in interphase cells, with some studies suggesting it also regulates gene expression (Yuen Science Adv 2017; Iwasaki Nature Comm 2019). Similar to Setd8, many subunits of the Condensin II complex are highly expressed in erythroid cells compared to most other cell types (biogps.org). We hypothesized that the Condensin II complex, in conjunction with Setd8 and H4K20me1, is important for establishing appropriate patterns of chromatin architecture and gene expression in maturing erythroblasts. To address this hypothesis, we deleted the NCAPH2 subunit in erythroid cells by crossing mice with floxed alleles of NCAPH2 with mice expressing cre-recombinase under the direction of the Erythropoietin receptor promotor (EpoRCre). Homozygous disruption of NCAPH2 was embryonic lethal by E13.5. NCAPH2 mutant embryos were similar in appearance to littermate controls until E12.5 when they developed notable pallor and a dramatic decline in the number of benzidine positive cells. Cell cycle analyses demonstrated that an accumulation of cells in G2/M preceded the dramatic decline in erythroblast numbers at E12.5. In contrast to cells from littermate controls, the NCAPH2 mutant cells were very heterogenous in cell and nuclear size and morphology. Surpisingly, most NCAPH2 mutant cells appeared to be hemoglobinized, suggesting sufficient iron accumulation and heme synthesis. In vitro cultures derived from primitive erythroid progenitors replicated in vivo findings, including normal early erythropoeisis, with significant abnormalities during mid- to late- maturation. Western blot in cycloheximide treated primitive erythroid cultures revealed that NCAPH2 has a long half-life, which likely contributes to the relative normalicy of early primitive erythoproesis. NCAPH2 mutant embryos also had a dramatic failure of definitive erythropoiesis, as evidenced by loss of mature erythroblasts in the fetal liver at E13.5. To gain insights into the mechanisms underlying these findings, we performed RNA-seq of NCAPH2 mutant, NCAPH2 het, and NCAPH2 WT erythroblasts from E11.5 embryos. Comparing NCAPH2 mutant and NCAPH2 WT erythroblasts there were 1121 down regulated genes and 743 upregulated genes (adj p-value &lt;0.05). As expected, the downregulated genes were enriched for pathways related to cell cycle, such as Mitotic Spindle Organization (adj pvalue 5e-42). The upregulated genes were enriched for a variety of pathways including p53 transcriptional network (adj pvalue 4e-10), neutrophil mediated immunity (2e-9), DNA-binding transcription factor (adj pvalue 7e-5), and regulation of erythrocyte differentiation (adj pvalue 5e-4). Intriguingly, 91/340 genes differentially expressed in Setd8 mutant erythroblats were also differentially expressed in NCAPH2 mutant cells, including genes typically repressed early in erythroid commitment, such as GATA2 and SPI1. Cut&Tag in CD34+ derived erythroblasts demonstrated occupancy of H4K20me1 at these loci. Mass spectrometry of proteins isolated via mono-mehtylated H4K20 peptides in erythroid extracts identified Condensin II components, supporting a model where the Condensin II complex directly interacts with H4K20me1. Together, these results demonstrate that the Condensin II complex is essential for erythropoiesis, and may work in conjunction with Setd8 and H4K20me1 to establish appropriate patterns of gene expression in maturing erythroblasts. Disclosures No relevant conflicts of interest to declare.

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