Eleven-nineteen Lysine-rich Leukemia Research Articles

Exploring novel protein biomarkers for early-stage diagnosis and prognosis of T-acute lymphoblastic leukemia (T-ALL)

BackgroundEfficient classification of T-acute lymphoblastic leukemia (T-ALL) involves considering various factors, such as age, white blood cell count, and chromosomal alterations. However, studying protein markers are crucial to improving T-ALL patients' diagnosis and treatment. A study analyzing the expression of proteomes was conducted to identify promising early-stage biomarkers for T-ALL patients MethodsLabel-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to analyze the blood proteins of both patients and healthy individuals to identify new biomarkers for T-ALL. The findings were validated by RT-PCR, ELISA and computational analysis ResultsThe study identified 1467 proteins in the blood, of which nine were upregulated and 35 were downregulated by more than 2-fold. T-ALL patients showed a significant increase in specific disease-related proteins, such as eleven-nineteen lysine-rich leukemia protein, triggering receptor expressed on myeloid cells 1, cisplatin resistance-associated-overexpressed protein, X-ray radiation resistance-associated protein 1, tumor necrosis factor receptor superfamily member 10D, protein S100-A8, and copine-4, by more than 3-fold ConclusionThe findings of this study provide a valuable protein map of leukemic cells and identify potential biomarkers for leukemic aggressiveness. However, further studies using larger T-ALL patient samples must confirm these preliminary results.

Zebrafish ELL-associated factors Eaf1/2 modulate erythropoiesis via regulating gata1a expression and WNT signaling to facilitate hypoxia tolerance

EAF1 and EAF2, the eleven-nineteen lysine-rich leukemia (ELL)-associated factors which can assemble to the super elongation complex (AFF1/4, AF9/ENL, ELL, and P-TEFb), are reported to participate in RNA polymerase II to actively regulate a variety of biological processes, including leukemia and embryogenesis, but whether and how EAF1/2 function in hematopoietic system related hypoxia tolerance during embryogenesis remains unclear. Here, we unveiled that deletion of EAF1/2 (eaf1−/− and eaf2−/−) caused reduction in hypoxia tolerance in zebrafish, leading to reduced erythropoiesis during hematopoietic processes. Meanwhile, eaf1−/− and eaf2−/− mutants showed significant reduction in the expression of key transcriptional regulators scl, lmo2, and gata1a in erythropoiesis at both 24 h post fertilization (hpf) and 72 hpf, with gata1a downregulated while scl and lmo2 upregulated at 14 hpf. Mechanistically, eaf1−/− and eaf2−/− mutants exhibited significant changes in the expression of epigenetic modified histones, with a significant increase in the binding enrichment of modified histone H3K27me3 in gata1a promoter rather than scl and lmo2 promoters. Additionally, eaf1−/− and eaf2−/− mutants exhibited a dynamic expression of canonical WNT/β-catenin signaling during erythropoiesis, with significant reduction in p-β-Catenin level and in the binding enrichment of both scl and lmo2 promoters with the WNT transcriptional factor TCF4 at 24 hpf. These findings demonstrate an important role of Eaf1/2 in erythropoiesis in zebrafish and may have shed some light on regeneration medicine for anemia and related diseases and on molecular basis for fish economic or productive traits, such as growth, disease resistance, hypoxia tolerance, and so on.

Sequence, structural and functional conservation among the human and fission yeast ELL and EAF transcription elongation factors.

Transcription elongation is a dynamic and tightly regulated step of gene expression in eukaryotic cells. Eleven nineteen Lysine rich Leukemia (ELL) and ELL Associated Factors (EAF) family of conserved proteins are required for efficient RNA polymerase II-mediated transcription elongation. Orthologs of these proteins have been identified in different organisms, including fission yeast and humans. In the present study, we have examined the sequence, structural and functional conservation between the fission yeast and human ELL and EAF orthologs. Our computational analysis revealed that these proteins share some sequence characteristics, and were predominantly disordered in both organisms. Our functional complementation assays revealed that both human ELL and EAF proteins could complement the lack of ell1+ or eaf1+ in Schizosaccharomyces pombe respectively. Furthermore, our domain mapping experiments demonstrated that both the amino and carboxyl terminal domains of human EAF proteins could functionally complement the S. pombe eaf1 deletion phenotypes. However, only the carboxyl-terminus domain of human ELL was able to partially rescue the phenotypes associated with lack of ell1+ in S. pombe. Collectively, our work adds ELL-EAF to the increasing list of human-yeast complementation gene pairs, wherein the simpler fission yeast can be used to further enhance our understanding of the role of these proteins in transcription elongation and human disease.

Arabidopsis thaliana possesses two novel ELL associated factor homologs.

Transcription elongation is one of the key steps at which RNA polymerase II-directed expression of protein-coding genes is regulated in eukaryotic cells. Different proteins have been shown to control this process, including the ELL/EAF family. ELL Associated Factors (EAFs) were first discovered in a yeast two-hybrid screen as interaction partners of the human ELL (Eleven nineteen Lysine-rich Leukemia) transcription elongation factor. Subsequently, they have been identified in different organisms, including Schizosaccharomyces pombe. However, no homolog(s) of EAF has as yet been characterized from plants. In the present work, we identified EAF orthologous sequences in different plants and have characterized two novel Arabidopsis thaliana EAF homologs, AtEAF-1 (At1g71080) and AtEAF-2 (At5g38050). Sequence analysis showed that both AtEAF-1 and AtEAF-2 exhibit similarity with its S. pombe EAF counterpart. Moreover, both Arabidopsis thaliana and S. pombe EAF orthologs share conserved sequence characteristic features. Computational tools also predicted a high degree of disorder in regions towards the carboxyl terminus of these EAF proteins. We demonstrate that AtEAF-2, but not AtEAF-1 functionally complements growth deficiencies of Schizosaccharomyces pombe eaf mutant. We also show that only AtEAF-1 displays transactivation potential resembling the S. pombe EAF ortholog. Subsequent expression analysis in A. thaliana showed that both homologs were expressed at varying levels during different developmental stages and in different tissues tested in the study. Individual null-mutants of either AtEAF-1 or AtEAF-2 are developmentally normal implying their functional redundancy. Taken together, our results provide first evidence that A. thaliana also possesses functional EAF proteins, suggesting an evolutionary conservation of these proteins across organisms.

Functional interaction between ELL transcription elongation factor and Epe1 reveals the role of Epe1 in the regulation of transcription outside heterochromatin.

Eleven-nineteen lysine-rich leukemia (ELL) is a eukaryotic RNA polymerase II transcription elongation factor. In Schizosaccharomyces pombe, it is important for survival under genotoxic stress conditions. However, the molecular basis underlying this function of ELL in S. pombe is yet to be deciphered. Here, we carried out a genetic screen to identify multicopy suppressor(s) that could restore normal growth of ell1 deletion mutant in the presence of DNA damaging agent. Sequence analysis of the identified suppressors revealed the anti-silencing protein, Epe1, as one of the suppressors of ell1 deletion associated genotoxic stress sensitivity. Our results further demonstrate that the overexpression of Epe1 could suppress all other phenotypes associated with the absence of Ell1. Moreover, transcriptional defect of ell1Δ strain could also be alleviated by the overexpression of Epe1. Epe1 also showed a physical interaction with Ell1. Interestingly, we also observed that the region of Epe1 encompassing 403-948 amino acids was indispensable for all the above functions. Furthermore, our results show that the overexpression of Epe1 causes increased H3K9 acetylation and RNA polymerase II recruitment. Taken together, our results show a functional interaction between Epe1 and Ell1, and this function is independent of the well-known JmjC and N-terminal transcriptional activation domains of Epe1 in S. pombe.

Linking Endoplasmic Reticular Stress and Alternative Splicing.

RNA splicing patterns in antibody-secreting cells are shaped by endoplasmic reticulum stress, ELL2 (eleven-nineteen lysine-rich leukemia gene 2) induction, and changes in the levels of snRNAs. Endoplasmic reticulum stress induces the unfolded protein response comprising a highly conserved set of genes crucial for cell survival; among these is Ire1, whose auto-phosphorylation drives it to acquire a regulated mRNA decay activity. The mRNA-modifying function of phosphorylated Ire1 non-canonically splices Xbp1 mRNA and yet degrades other cellular mRNAs with related motifs. Naïve splenic B cells will activate Ire1 phosphorylation early on after lipopolysaccharide (LPS) stimulation, within 18 h; large-scale changes in mRNA content and splicing patterns result. Inhibition of the mRNA-degradation function of Ire1 is correlated with further differences in the splicing patterns and a reduction in the mRNA factors for snRNA transcription. Some of the >4000 splicing changes seen at 18 h after LPS stimulation persist into the late stages of antibody secretion, up to 72 h. Meanwhile some early splicing changes are supplanted by new splicing changes introduced by the up-regulation of ELL2, a transcription elongation factor. ELL2 is necessary for immunoglobulin secretion and does this by changing mRNA processing patterns of immunoglobulin heavy chain and >5000 other genes.

The amino-terminal domain of ELL transcription elongation factor is essential for ELL function in Schizosaccharomyces pombe.

Transcriptional elongation is a critical step for regulating expression of protein-coding genes. Multiple transcription elongation factors have been identified in vitro, but the physiological roles of many of them are still not clearly understood. The ELL (Eleven nineteen Lysine rich Leukemia) family of transcription elongation factors are conserved from fission yeast to humans. Schizosaccharomyces pombe contains a single ELL homolog (SpELL) that is not essential for its survival. Therefore to gain insights into the in vivo cellular functions of SpELL, we identified phenotypes associated with deletion of ell1 in S. pombe. Our results demonstrate that SpELL is required for normal growth of S. pombe cells. Furthermore, cells lacking ell1+ exhibit a decrease in survival when exposed to DNA-damaging conditions, but their growth is not affected under environmental stress conditions. ELL orthologs in different organisms contain three conserved domains, an amino-terminal domain, a middle domain and a carboxyl-terminal domain. We also carried out an in vivo functional mapping of these conserved domains within S. pombe ELL and uncovered a critical role for its amino-terminus in regulating all its cellular functions, including growth under different conditions, transcriptional elongation potential and interaction with S. pombe EAF. Taken together our results suggest that the domain organization of ELL proteins is conserved across species, but the in vivo functions as well as the relationship between the various domains and roles of ELL show species-specific differences.

Structure function characterization of the ELL Associated Factor (EAF) from Schizosaccharomyces pombe

EAF (ELL Associated Factor) proteins interact with the transcription elongation factor, ELL (Eleven nineteen Lysine rich Leukemia) and enhance its ability to stimulate RNA polymerase II-mediated transcriptional elongation in vitro. Schizosaccharomyces pombe contains a single homolog of EAF (SpEAF), which is not essential for survival of S. pombe in contrast to its essential higher eukaryotic homologs. The physiological role of SpEAF is not well understood. In this study, we show that S. pombe EAF is important in regulating growth of S. pombe cells during normal growth conditions. Moreover, SpEAF is also essential for survival under conditions of DNA damage, while its deletion does not affect growth under environmental stress conditions. Our in vivo structure-function studies further demonstrate that while both the amino and carboxyl terminal domains of SpEAF possess the potential to activate transcription, only the amino terminal domain of SpEAF is involved in interaction with the S. pombe ELL protein. The carboxyl-terminus of SpEAF is required for rescue of the growth defect under normal and DNA damaging conditions that is associated with the absence of SpEAF. Using bioinformatics and circular dichroism spectroscopy, we show that the carboxyl-terminus of SpEAF has a disordered conformation. Furthermore, addition of trifluoroethanol triggered its transition from a disordered to α-helical conformation. Taken together, the results presented here identify novel structural and functional features of SpEAF protein, providing insights into how EAF proteins may enforce transcriptional control of gene expression.

073_16735-H5 Reverse Electrical Remodeling Induced by Sinus Rhythm and Calcium Channel Blockers May Play a Role for Better Long-Term Follow-Up Results in Patients With Long Standing Persistent Atrial Fibrillation and Treated With Cryo-Balloon Ablation

To characterize the functions of MLL fusion transcripts, we cloned the gene that fuses to MLL in the translocation t(11;19)(q23;p13.1). This translocation is distinct from another type of 11;19 translocation with a 19p13.3 breakpoint that results in the fusion of MLL to the ENL gene. By PCR screening of a cDNA library prepared from a patient9s leukemia cells with this translocation, we obtained a fusion transcript containing exon 7 of MLL and sequence of an unknown gene. The sequence of this gene was amplified and used as a probe to screen a fetal brain cDNA library. On Northern blot analysis, this cDNA detected a 4.4-kb transcript that was abundant in peripheral blood leukocytes, skeletal muscle, placenta, and testis and expressed at lower levels in spleen, thymus, heart, brain, lung, kidney, liver, and ovary. In addition, a 2.8-kb transcript was present in peripheral blood, testis, and placenta. On zoo blots, this gene was shown to be evolutionarily conserved in 10 mammalian species as well as in chicken, frog, and fish. We have named this gene ELL (for eleven-nineteen lysine-rich leukemia gene). A highly basic, lysine-rich motif of the predicted ELL protein is homologous to similar regions of several proteins, including the DNA-binding domain of poly(ADP-ribose) polymerase. The characterization of the normal functions of ELL as well as its altered function when fused to MLL will be critical to further our understanding of the mechanisms of leukemogenesis.

Selective expression of the transcription elongation factor ELL3 in B cells prior to ELL2 drives proliferation and survival

B cell activation is dependent on a large increase in transcriptional output followed by focused expression on secreted immunoglobulin as the cell transitions to an antibody producing plasma cell. The rapid transcriptional induction is facilitated by the release of poised RNA pol II into productive elongation through assembly of the super elongation complex (SEC). We report that a SEC component, the Eleven -nineteen Lysine-rich leukemia (ELL) family member 3 (ELL3) is dynamically up-regulated in mature and activated human B cells followed by suppression as B cells transition to plasma cells in part mediated by the transcription repressor PRDM1. Burkitt’s lymphoma and a sub-set of Diffuse Large B cell lymphoma cell lines abundantly express ELL3. Depletion of ELL3 in the germinal center derived lymphomas results in severe disruption of DNA replication and cell division along with increased DNA damage and cell death. This restricted utilization and survival dependence reveal a key step in B cell activation and indicate a potential therapeutic target against B cell lymphoma’s with a germinal center origin.

The effect of eleven-nineteen lysine-rich leukemia gene 2 on proliferation of prostate cancer cell C4-2

Objective To observe the level of eleven-nineteen lysine-rich leukemia gene 2 (ELL2) in human prostate cancer tissues and its effect on proliferation of prostate cancer cell C4-2. Methods The expression of ELL2 in human prostate cancer tissues was analyzed by immunohistochemical method, and the relationship between ELL2 and malignancy degree of prostate cancer was evaluated. The human prostate cancer cell line C4-2 was stimulated with synthetic androgen R1881 to detect its response to androgen at different time points. ELL2-small interfering RNA (siRNA) and overexpression of ELL2-bearing plasmid were transfected into C4-2 cells respectively, then cell proliferation was evaluated by methyl thiazol tetrazolium (MTT) assay. The DNA synthesis ability of cells was assessed by 5-Bromo-2’-deoxy-uridine (BrdU) incorporation assay. Results The expression of ELL2 was significantly lower in human prostate cancer than that of benign specimen, and decreased with the increase of malignancy of prostate cancer. The correlation analysis between ELL2 and prostate cancer Gleason score reveal a negative correlation result (r=0.836, P=0.000). The protein expression of ELL2 was significantly increased in 48 h and increased by (38.27±10.11)% (t=7.573, P=0.005) and (58.34±13.70)% (t=8.519, P=0.003) at 24 h and 48 h, respectively. The expression of ELL2 protein in the two RNA interference groups were significantly decreased, and the decreased rate were (56.38±11.26)% (t=10.010, P=0.002) and (65.27±12.10)% (t=10.790, P=0.002), respectively. While the ELL2 protein expression in the overexpression group was significantly increased at (80.23±20.94)% (t=7.662, P=0.005). The proliferation ability of C4-2 cells was significantly increased (39.28±8.52)% (t=11.300, P=0.000) and (48.2±7.87)% (t=15.000, P=0.000) in the two RNA interference groups, respectively, compared with the control group. While its proliferation ability decreased (42.74±10.38)% (t=10.090, P=0.000) in ELL2-bearing plasmid. Transfection of ELL2-specific interfering RNA increased the ability of DNA synthesis, while the transfection of ELL2-bearing plasmid cells had lower DNA synthesis ability. Conclusion ELL2 is lowly expressed in human prostate cancer tissues and negatively correlated with the malignant degree. ELL2 can inhibit the proliferation of prostate cancer cells to a certain extent. Key words: Prostate cancer; Eleven-nineteen lysine-rich leukemia gene 2; Proliferation

The Tax oncogene enhances ELL incorporation into p300 and P-TEFb containing protein complexes to activate transcription

The eleven-nineteen lysine-rich leukemia protein (ELL) is a key regulator of RNA polymerase II mediated transcription. ELL facilitates RNA polymerase II transcription pause site entry and release by dynamically interacting with p300 and the positive transcription elongation factor b (P-TEFb). In this study, we investigated the role of ELL during the HTLV-1 Tax oncogene induced transactivation. We show that ectopic expression of Tax enhances ELL incorporation into p300 and P-TEFb containing transcriptional complexes and the subsequent recruitment of these complexes to target genes in vivo. Depletion of ELL abrogates Tax induced transactivation of the immediate early genes Fos, Egr2 and NF-kB, suggesting that ELL is an essential cellular cofactor of the Tax oncogene. Thus, our study identifies a novel mechanism of ELL-dependent transactivation of immediate early genes by Tax and provides the rational for further defining the genome-wide targets of Tax and ELL.

Abstract 2331: Transcriptional repressor PRDM1/Blimp-1 directly regulates transcriptional elongation factor ELL3 during terminal B-cell differentiation

Abstract As the master regulator of B-cell differentiation, PRDM1 is well known for its ability to extinguish a network of transcription factors essential in maintaining the B-cell phenotype. In addition PRDM1 is a tumor suppressor in Diffuse Large B-Cell Lymphoma (DLBCL). However its molecular targets remain to be clearly elucidated. For that purpose we have identified novel targets of PRDM1 by chromatin immunoprecipitation (ChIP) and deep sequencing. One of the strongest associations with PRDM1 was observed at the Eleven-Nineteen Lysine-rich Leukemia (ELL) 3 promoter. The ELL family of transcriptional elongation factors (ELL, ELL2, and ELL3) is reported to increase the catalytic rate of RNA polymerase (pol) II transcription through assembly of the Super Elongation Complex (SEC), although their role in activated B-cells is unknown. Two potential consensus PRDM1 binding sites were mapped within the ELL3 proximal promoter and endogenous PRDM1 binding was confirmed by direct ChIP-qPCR analysis in multiple cell lines. The cloned ELL3 promoter was highly active in B-cell lines and co-transfection with PRDM1 significantly repressed activity. Mutation of both PRDM1 binding sites eliminated repression, while mutation of either individual site resulted in a partial loss of repression. To establish the expression pattern of ELL family members in normal human B-cells, primary naïve B-cells were isolated by negative selection and either mildly activated with IL-2 and IL-4 or differentiated in the presence of IL-2, IL-21, anti-IgM, and CD40 cross-linking antibody. Naïve B-cells did not express detectable protein levels of any ELL family members. Mild activation induced the expression of ELL and ELL3 while in contrast differentiation selectively suppressed ELL3 while inducing ELL2 and PRDM1. Expression of ELL, ELL2, and ELL3 was further profiled in a panel of B-cell lymphomas and multiple myeloma cell lines. Cell lines representing mature activated B-cells (Burkitts, DLBCL) displayed robust expression of ELL3 while lines of a pre B-cell, naïve B-cell or differentiated plasma cell did not express ELL3. ELL2 expression was observed only in the plasma cell lines, consistent with previous reports. ELL was ubiquitously expressed at low levels. shRNA knockdown of ELL3 in ELL3 expressing B-cell lymphomas reveals that ELL3 is required for cell proliferation. Together these results establish ELL3 as an activated B-cell restricted elongation factor and suggests that a switch from ELL3 to ELL2 occurs during terminal differentiation. Dependence on ELL3 for proliferation of lymphoma lines suggests it may be a valid therapeutic target in ELL3 high expressing tumors and is consistent with suppression by the tumor suppressor PRDM1. Citation Format: Lou-Ella M.M. Alexander, January M. Watters, Michelle Maurin, Kenneth L. Wright. Transcriptional repressor PRDM1/Blimp-1 directly regulates transcriptional elongation factor ELL3 during terminal B-cell differentiation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2331. doi:10.1158/1538-7445.AM2014-2331

Abstract 1349: Chemosensitization of breast cancer cell lines by Ell3

Abstract Ell3 is a RNA polymerase II transcription elongation factor that is enriched in testis. The C-terminal domain of Ell3 shows strong similarities to that of Ell (eleven-nineteen lysine-rich leukemia gene), which acts as a negative regulator of p53. Recent studies in our laboratory showed that Ell3 induces the differentiation of mouse embryonic stem cells by protecting differentiating cells from apoptosis by the promotion of p53 degradation. In this study, we evaluated the function of Ell3 in breast cancer cell lines. Contrary to our expectation, ectopic expression of Ell3 in MCF7 induces CDDP (cisplatin) susceptibility by enhancing p53 dependent apoptosis. CDDP treatments leads to the accumulation of p53 protein in the Ell3 overexpressing MCF7. We confirmed that Ell3 inhibits proteosomal degradation of p53 upon cisplatin treatment in MCF7. Ell3 also confer chemosensitivity to MCF7 to DNA damaging agent, camptothecin. The treatment of camptothecin to Ell3 overexpressing MCF7 results in the accumulation of cells at G2/M phase of cell cycle by the increases of p53 protein. Consistent with these results, Ell3 downregulates the expression of MDM2, which binds p53 and marks it for degradation using the proteasome. The underlying mechanism of Ell3 to regulate MDM2 expression is under investigation. Citation Format: Hee-Jin Ahn, Sun-Hee Heo, Gwangil Kim, Kyung-Soon Park. Chemosensitization of breast cancer cell lines by Ell3. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1349. doi:10.1158/1538-7445.AM2014-1349

ELL Inhibits E2F1 Transcriptional Activity by Enhancing E2F1 Deacetylation via Recruitment of Histone Deacetylase 1

ELL (eleven-nineteen lysine-rich leukemia protein) was first identified as a translocation partner of MLL in acute myeloid leukemia; however, the exact mechanism of its action has remained elusive. In this study, we identified ELL as a direct downstream target gene of E2F1. Coimmunoprecipitation assays showed that ELL interacted with E2F1 in vitro and in vivo, leading to inhibition of E2F1 transcriptional activity. In addition, ELL enhanced E2F1 deacetylation via recruitment of histone deacetylase 1 (HDAC1). Notably, the MLL-ELL fusion protein lost the inhibitory role of ELL in E2F1 transcriptional activity. Furthermore, DNA damage induced ELL in an E2F1-dependent manner and ELL protected cells against E2F1-dependent apoptosis. Our findings not only connect ELL to E2F1 function and uncover a novel role of ELL in response to DNA damage but also provide an insight into the mechanism for MLL-ELL-associated leukemogenesis.

ELL, a novel TFIIH partner, is involved in transcription restart after DNA repair

DNA lesions that block transcription may cause cell death even when repaired, if transcription does not restart to reestablish cellular metabolism. However, transcription resumption after individual DNA-lesion repair remains poorly described in mechanistic terms and its players are largely unknown. The general transcription factor II H (TFIIH) is a major actor of both nucleotide excision repair subpathways of which transcription-coupled repair highlights the interplay between DNA repair and transcription. Using an unbiased proteomic approach, we have identified the protein eleven-nineteen lysine-rich leukemia (ELL) as a TFIIH partner. Here we show that ELL is recruited to UV-damaged chromatin in a Cdk7- dependent manner (a component of the cyclin-dependent activating kinase subcomplex of TFIIH). We demonstrate that depletion of ELL strongly hinders RNA polymerase II (RNA Pol II) transcription resumption after lesion removal and DNA gap filling. Lack of ELL was also observed to increase RNA Pol II retention to the chromatin during this process. Identifying ELL as an essential player for RNA Pol II restart during cellular DNA damage response opens the way to obtaining a mechanistic description of transcription resumption after DNA repair.

The RNA Pol II Elongation Factor Ell3 Marks Enhancers in ES Cells and Primes Future Gene Activation

Enhancers play a central role in precisely regulating the expression of developmentally regulated genes. However, the machineries required for enhancer-promoter communication have remained largely unknown. We have found that Ell3, a member of the Ell (eleven-nineteen lysine-rich leukemia gene) family of RNA Pol II elongation factors, occupies enhancers in embryonic stem cells. Ell3's association with enhancers is required for setting up proper Pol II occupancy at the promoter-proximal regions of developmentally regulated genes and for the recruitment of the super elongation complex (SEC) to these loci following differentiation signals. Furthermore, Ell3 binding to inactive or poised enhancers is essential for stem cell specification. We have also detected the presence of Pol II and Ell3 in germ cell nuclei. These findings raise the possibility that transcription factors could prime gene expression by marking enhancers in ES cells or even as early as in the germ cell state.

Ribavirin-Induced Intracellular GTP Depletion Upregulates Factor VII Expression

AbstractAbstract 1113In this study, we investigated the molecular basis of upregulation of factor VII (FVII) gene expression by ribavirin, and found that intracellular GTP depletion induced by ribavirin activated FVII gene transcription and modulated transcription elongation.In 2006, Yamamoto et al. reported that anti-hepatitis C virus (HCV) agent ribavirin elevated the activity of FVII in HCV-infected hemophilia patients; however, the precise mechanisms were still unknown. In addition, the anti-HCV mechanisms of ribavirin were not yet fully elucidated, although the extended studies have been done. We investigated the effects of ribavirin in vitro and confirmed the approximately 4-fold upregulation of FVII mRNA by ribavirin treatment in HepG2 cells. FVII mRNA was increased in a dose-dependent manner up to 100μg/mL of ribavirin at a lower concentration than therapeutic concentration of 150μg/mL. FVII mRNA induction by ribavirin was also observed in a time-dependent manner from 24 h to 72 h after treatment.Ribavirin metabolite ribavirin 5'-monophosphate is one of the IMP dehydrogenase (IMPDH) inhibitors, and the other IMPDH inhibitors mycophenolic acid (MPA) and 6-mercaptupurine (6-MP) also induced FVII upregulation. It is well known that inhibition of IMPDH causes intracellular GTP depletion, and guanosine supplementation to salvage GTP could reverse FVII mRNA increase in ribavirin-treated cells. These results indicated that cellular GTP reduction associated with FVII gene upregulation.The mechanisms of gene upregulation by GTP depletion were not elucidated. The promoter activities and mRNA stability of FVII were analyzed under ribavirin treatment. The FVII gene promoter activity was enhanced up to 1.5-fold by ribavirin treatment; however the activation did not reach 4-fold induction of FVII mRNA increase. There was no significant change of FVII mRNA half-life in ribavirin-treated cells. Since the promoter activation might display transcription initiation capacity, the contribution of transcription elongation stage was further investigated.Transcription elongation was regulated by phosphorylation of carbo-terminal domain (CTD) of RNA polymerase II (PolII). Transcription elongation factor P-TEFb (positive-transcription elongation factor b), which consists as a complex of CDK9 and cyclin T, phosphorylates Ser of PolII CTD. The kinase activity of P-TEFb could be inhibited by 5,6-dichlorobenzimidazole 1-b-D-ribofuranoside (DRB). In FVII gene upregulation, DRB completely canceled ribavirin-induced FVII mRNA increase. We also performed nuclear run-on assay to verify the potential transcription elongation capacity of paused PolII, and observed a dramatic increase of FVII mRNA in ribavirin-treated cells. These results suggested that ribavirin-induced FVII gene upregulation was caused not only by transcription initiation but also by accelerated transcription elongation rate.There are various transcription factor associated with transcription elongation in addition to P-TEFb, such as elongin, ELL (eleven nineteen-lysine rich leukemia). We found that ELL3, a member of ELL family protein, was upregulated by ribavirin treatment. A ELL3 mRNA increase occurred prior to FVII mRNA upregulation, and the ELL3 upregulation was also canceled by guanosine supplementation. These results indicated ELL3 induction by ribavirin was also a response to cellular GTP depletion. To confirm the contribution of ELL3 protein to FVII gene transcription elongation, we used siRNAs specific to ELL3 and as expected, knockdown of ELL3 resulted in diminished FVII upregulation. A chromatin immunoprecipitation (ChIP) revealed ELL3 recruitment to the FVII gene, and the recruitments of PolII and CDK9 were also enhanced by ribavirin treatment.Taken together, FVII gene upregulation by ribavirin was associated with intracellular GTP depletion. The GTP reduction mainly modulates transcription elongation rate rather than transcription initiation, though the relationships between cellular GTP depletion and enhanced transcription elongation must be investigated. This study uncovered candidate mechanisms of ribavirin and the other IMPDH inhibitors and highlights a development of novel pharmaceutical therapies for hemophilia. Disclosures:No relevant conflicts of interest to declare.

Determination of protein-protein interactions of human eleven-nineteen lysine-rich leukemia protein ELL2 (176.8)

Abstract Eleven-nineteen lysine-rich leukemia (ELL) genes produce protein factors that aid in transcription elongation. ELL1 was originally found as a fusion partner with MLL in some multiple lineage leukemias. ELL2 mRNA and protein are induced 6-fold in plasma cells. ELL1 and ELL2 are very similar except for 2 regions between amino acids 352 - 443 and 473 - 516, indicating that they may have different sets of interacting proteins. Yeast two-hybrid (Y2H) assays were performed to determine what proteins might interact with each ELL. ELL2, was subcloned into four main sections (A1, A2, B, and C) to determine specific locations of interaction. The C portion of ELL2, aa 516 to 640, autoactivated transcription in the Y2H and was not further analyzed. Meanwhile, the A1 region interacted with CEP192 (regulator of pericentriolar material recruitment, centrosome maturation, and centriole duplication in mammalian cells); the A2 region interacted with RP3 gene (XLRP gene 3), FIGNLI (new subfamily member of ATPases associated with diverse cellular activities); and the B region interacted with HIFIA (transcription factor found in mammalian cells under reduced oxygen tension that plays an essential role in hypoxia) and SEC63 (a translocation protein). Further mapping of the interactions of the ELLs may help us to clarify their functions in gene regulation and plasma cell development.

The Little Elongation Complex Regulates Small Nuclear RNA Transcription

Eleven-nineteen lysine-rich leukemia (ELL) participates in the super elongation complex (SEC) with the RNA polymerase II (Pol II) CTD kinase P-TEFb. SEC is a key regulator in the expression of HOX genes in mixed lineage leukemia (MLL)-based hematological malignancies, in the control of induced gene expression early in development, and in immediate early gene transcription. Here, we identify an SEC-like complex in Drosophila, as well as a distinct ELL-containing complex that lacks P-TEFb and other components of SEC named the "little elongation complex" (LEC). LEC subunits are highly enriched at RNA Pol II-transcribed small nuclear RNA (snRNA) genes, and the loss of LEC results in decreased snRNA expression in both flies and mammals. The specialization of the SEC and LEC complexes for mRNA and snRNA-containing genes, respectively, suggests the presence of specific classes of elongation factors for each class of genes transcribed by RNA polymerase II.