Subject(s)
CRISPR-Cas Systems/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Leukemia/genetics , Osteonectin/genetics , Animals , Disease Progression , Gene Editing/methods , Hematologic Neoplasms/genetics , Hematologic Neoplasms/pathology , Heterografts , Humans , Leukemia/pathology , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Tumor Microenvironment/geneticsABSTRACT
We conducted a cytogenetic analysis of 642 children with de novo acute myeloid leukemia (AML) treated on the AML-Berlin-Frankfurt-Münster (BFM) 04 protocol to determine the prognostic value of specific chromosomal aberrations including monosomal (MK+), complex (CK+) and hypodiploid (HK+) karyotypes, individually and in combination. Multivariate regression analysis identified in particular MK+ (n=22) as a new independent risk factor for poor event-free survival (EFS 23±9% vs 53±2% for all other patients, P=0.0003), even after exclusion of four patients with monosomy 7 (EFS 28±11%, P=0.0081). CK+ patients without MK had a better prognosis (n=47, EFS 47±8%, P=0.46) than those with MK+ (n=12, EFS 25±13%, P=0.024). HK+ (n=37, EFS 44±8% for total cohort, P=0.3) influenced outcome only when t(8;21) patients were excluded (remaining n=16, EFS 9±8%, P<0.0001). An extremely poor outcome was observed for MK+/HK+ patients (n=10, EFS 10±10%, P<0.0001). Finally, isolated trisomy 8 was also associated with low EFS (n=16, EFS 25±11%, P=0.0091). In conclusion, monosomal karyotype is a strong and independent predictor for high-risk pediatric AML. In addition, isolated trisomy 8 and hypodiploidy without t(8;21) coincide with dismal outcome. These results have important implications for risk stratification and should be further validated in independent pediatric cohorts.
Subject(s)
Genetic Variation , Genotype , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/mortality , Chromosome Aberrations , Chromosomes, Human, Pair 7 , Clinical Trials as Topic , Female , Humans , Karyotype , Leukemia, Myeloid, Acute/diagnosis , Male , Monosomy , Mutation , Prognosis , Survival AnalysisABSTRACT
Inhibition of anti-apoptotic BCL-2 (B-cell lymphoma 2) has recently emerged as a promising new therapeutic strategy for the treatment of a variety of human cancers, including leukemia. Here, we used T-cell acute lymphoblastic leukemia (T-ALL) as a model system to identify novel synergistic drug combinations with the BH3 mimetic venetoclax (ABT-199). In vitro drug screening in primary leukemia specimens that were derived from patients with high risk of relapse or relapse and cell lines revealed synergistic activity between venetoclax and the BET (bromodomain and extraterminal) bromodomain inhibitor JQ1. Notably, this drug synergism was confirmed in vivo using T-ALL cell line and patient-derived xenograft models. Moreover, the therapeutic benefit of this drug combination might, at least in part, be mediated by an acute induction of the pro-apoptotic factor BCL2L11 and concomitant reduction of BCL-2 upon BET bromodomain inhibition, ultimately resulting in an enhanced binding of BIM (encoded by BCL2L11) to BCL-2. Altogether, our work provides a rationale to develop a new type of targeted combination therapy for selected subgroups of high-risk leukemia patients.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Azepines/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Molecular Targeted Therapy , Neoplasm Proteins/antagonists & inhibitors , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Sulfonamides/pharmacology , Triazoles/pharmacology , Animals , Azepines/administration & dosage , Bcl-2-Like Protein 11/biosynthesis , Bcl-2-Like Protein 11/genetics , Bridged Bicyclo Compounds, Heterocyclic/administration & dosage , Cell Cycle Proteins , Cell Line, Tumor , Drug Synergism , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Nuclear Proteins/antagonists & inhibitors , Protein Domains , Sulfonamides/administration & dosage , Transcription Factors/antagonists & inhibitors , Triazoles/administration & dosage , Xenograft Model Antitumor AssaysABSTRACT
Chromosomal rearrangements of the mixed lineage leukemia (MLL/KMT2A) gene leading to oncogenic MLL-fusion proteins occur in ~10% of acute leukemias and are associated with poor clinical outcomes, emphasizing the need for new treatment modalities. Inhibition of the DOT1-like histone H3K79 methyltransferase (DOT1L) is a specific therapeutic approach for such leukemias that is currently being tested in clinical trials. However, in most MLL-rearranged leukemia models responses to DOT1L inhibitors are limited. Here, we performed deep-coverage short hairpin RNA sensitizer screens in DOT1L inhibitor-treated MLL-rearranged leukemia cell lines and discovered that targeting additional nodes of MLL complexes concomitantly with DOT1L inhibition bears great potential for superior therapeutic results. Most notably, combination of a DOT1L inhibitor with an inhibitor of the MLL-Menin interaction markedly enhanced induction of differentiation and cell killing in various MLL disease models including primary leukemia cells, while sparing normal hematopoiesis and leukemias without MLL rearrangements. Gene expression analysis on human and murine leukemic cells revealed that target genes of MLL-fusion proteins and MYC were suppressed more profoundly upon combination treatment. Our findings provide a strong rationale for a novel targeted combination therapy that is expected to improve therapeutic outcomes in patients with MLL-rearranged leukemia.
Subject(s)
Drug Resistance, Neoplasm/genetics , Enzyme Inhibitors/pharmacology , Gene Rearrangement , Histone-Lysine N-Methyltransferase/genetics , Leukemia/drug therapy , Methyltransferases/metabolism , Myeloid-Lymphoid Leukemia Protein/genetics , Proto-Oncogene Proteins/antagonists & inhibitors , RNA, Small Interfering/genetics , Animals , Apoptosis , Cell Proliferation , Drug Resistance, Neoplasm/drug effects , Female , Gene Expression Regulation, Leukemic , Histone-Lysine N-Methyltransferase/metabolism , Humans , Leukemia/genetics , Leukemia/pathology , Methyltransferases/genetics , Mice , Mice, Inbred C57BL , Mice, Nude , Myeloid-Lymphoid Leukemia Protein/metabolism , Tumor Cells, CulturedABSTRACT
BACKGROUND: The outcome in children and adolescents with high-risk (HR) acute myeloid leukemia (AML) is still unsatisfactory. Therefore, in study AML-BFM 2004 we aimed to improve outcome of HR-patients by adding moderately dosed 2-Chloro-2-Deoxyadenosine (2-CDA) to the respective consolidation treatment backbone without increasing toxicity. The aim was to improve prognosis especially in FAB M4/M5/MLL patients, who represent the largest subgroup of HR patients. PATIENTS AND METHODS: In total, 343 children and adolescents with HR-AML were randomized to receive or not 2-CDA (6 mg/m²/d, days 1, 3) in combination with cytarabine/idarubicine (AI=500 mg/m² cytarabine 5 days continuous infusion plus 7 mg/m²/d idarubicin, days 3 and 5). RESULTS: RESULTS for patients of the AI/2-CDA arm (n=168) vs. the AI-arm (n=175) were similar: 5-year overall survival 68±4 vs. 72±4%, plogrank=0.38, event-free survival 53±4 vs. 49±4%, plogrank=0.77; cumulative incidence of relapse at 5 years: 35±4 vs. 37±4%, p(Gray)=0.89. RESULTS in patients with MLL rearrangement or FAB M4/M5 were also similar in the treatment groups. In addition, toxicities did not differ between the two arms. CONCLUSION: We conclude that additional, moderate dose 2-CDA does not improve prognosis in HR-patients when given during consolidation treatment. Its effect might be too low in this multidrug regimen, where the strongest effects are achieved during induction, or the chosen dose of 2-CDA might have been too low.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cladribine/administration & dosage , Cladribine/adverse effects , Leukemia, Myeloid, Acute/drug therapy , Adolescent , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Child , Child, Preschool , Cytarabine/administration & dosage , Cytarabine/adverse effects , Dose-Response Relationship, Drug , Drug Administration Schedule , Humans , Idarubicin/administration & dosage , Idarubicin/adverse effects , Infusions, Intravenous , Leukemia, Myeloid, Acute/diagnosis , Leukemia, Myeloid, Acute/mortality , Prognosis , Survival RateABSTRACT
The prognosis for children with high-risk relapsed acute lymphoblastic leukemia (ALL) is poor. Here, we assessed the prognostic importance of response during induction and consolidation treatment prior to hematopoietic stem cell transplantation (HSCT) aiming to evaluate the best time to assess minimal residual disease (MRD) for intervention strategies and in future trials in high-risk ALL relapse patients. Included patients (n=125) were treated uniformly according to the ALL-REZ BFM (Berlin-Frankfurt-Münster) 2002 relapse trial (median follow-up time=4.8 years). Patients with MRD ⩾10(-3) after induction treatment (76/119, 64%) or immediately preceding HSCT (19/71, 27%) had a significantly worse probability of disease-free survival 10 years after relapse treatment begin, with 26% (±6%) or 23% (±7%), respectively, compared with 58% (±8%) or 48% (±7%) for patients with MRD <10(-3). Conventional intensive consolidation treatment reduced MRD to <10(-3) before HSCT in 63% of patients, whereas MRD remained high or increased in the rest of this patient group. Our data support that MRD after induction treatment can be used to quantify the activity of different induction treatment strategies in phase II trials. MRD persistence at ⩾10(-3) before HSCT reflects a disease highly resistant to conventional intensive chemotherapy and requiring prospective controlled investigation of new treatment strategies and drugs.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Monitoring, Physiologic , Neoplasm Recurrence, Local/drug therapy , Neoplasm, Residual/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Adolescent , Child , Female , Follow-Up Studies , Hematopoietic Stem Cell Transplantation , Humans , Induction Chemotherapy , Male , Neoplasm Recurrence, Local/mortality , Neoplasm Recurrence, Local/pathology , Neoplasm Staging , Neoplasm, Residual/mortality , Neoplasm, Residual/pathology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/mortality , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Prognosis , Prospective Studies , Risk Factors , Survival RateABSTRACT
In adult therapy, arsenic trioxide (ATO) and all-trans-retinoic acid (ATRA) are recognized as active treatment of relapsed acute promyelocytic leukemia (APL). The efficacy of this combination in pediatric APL has not yet been well established. We report the case of a 6-year-old girl with relapsed APL, with a PML-RARα mutation, treated with a combination of ATO and ATRA. Over a period of 5 months, she received in total, 75 doses of intravenous ATO and 40 doses of oral ATRA. Currently, 22 months after relapse, she is still in complete remission. Here, we describe treatment of a relapsed APL in a child with limited treatment of ATO and ATRA and review the literature.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Leukemia, Promyelocytic, Acute/drug therapy , Arsenic Trioxide , Arsenicals/administration & dosage , Child , Female , Humans , Oxides/administration & dosage , Recurrence , Tretinoin/administration & dosageABSTRACT
Histone deacetylase (HDAC) inhibitors (HDACis) are well-characterized anti-cancer agents with promising results in clinical trials. However, mechanistically little is known regarding their selectivity in killing malignant cells while sparing normal cells. Gene expression-based chemical genomics identified HDACis as being particularly potent against Down syndrome-associated myeloid leukemia (DS-AMKL) blasts. Investigating the antileukemic function of HDACis revealed their transcriptional and post-translational regulation of key autophagic proteins, including ATG7. This leads to suppression of autophagy, a lysosomal degradation process that can protect cells against damaged or unnecessary organelles and protein aggregates. DS-AMKL cells exhibit low baseline autophagy due to mammalian target of rapamycin (mTOR) activation. Consequently, HDAC inhibition repressed autophagy below a critical threshold, which resulted in accumulation of mitochondria, production of reactive oxygen species, DNA damage and apoptosis. Those HDACi-mediated effects could be reverted upon autophagy activation or aggravated upon further pharmacological or genetic inhibition. Our findings were further extended to other major acute myeloid leukemia subgroups with low basal level autophagy. The constitutive suppression of autophagy due to mTOR activation represents an inherent difference between cancer and normal cells. Thus, via autophagy suppression, HDACis deprive cells of an essential pro-survival mechanism, which translates into an attractive strategy to specifically target cancer cells.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Autophagy/drug effects , Histone Deacetylase Inhibitors/pharmacology , Leukemia, Myeloid/pathology , Animals , Humans , Leukemia, Myeloid/immunology , Leukemia, Myeloid/metabolism , Mice , Reactive Oxygen Species/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Intrachromosomal amplification of chromosome 21 (iAMP21) defines a distinct cytogenetic subgroup of childhood B-cell precursor acute lymphoblastic leukaemia (BCP-ALL). To date, fluorescence in situ hybridisation (FISH), with probes specific for the RUNX1 gene, provides the only reliable detection method (five or more RUNX1 signals per cell). Patients with iAMP21 are older (median age 9 years) with a low white cell count. Previously, we demonstrated a high relapse risk when these patients were treated as standard risk. Recent studies have shown improved outcome on intensive therapy. In view of these treatment implications, accurate identification is essential. Here we have studied the cytogenetics and outcome of 530 iAMP21 patients that highlighted the association of specific secondary chromosomal and genetic changes with iAMP21 to assist in diagnosis, including the gain of chromosome X, loss or deletion of chromosome 7, ETV6 and RB1 deletions. These iAMP21 patients when treated as high risk showed the same improved outcome as those in trial-based studies regardless of the backbone chemotherapy regimen given. This study reinforces the importance of intensified treatment to reduce the risk of relapse in iAMP21 patients. This now well-defined patient subgroup should be recognised by World Health Organisation (WHO) as a distinct entity of BCP-ALL.
Subject(s)
Chromosomes, Human, Pair 21 , Cytogenetic Analysis , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Adolescent , Adult , Child , Child, Preschool , Core Binding Factor Alpha 2 Subunit/genetics , Female , Humans , In Situ Hybridization, Fluorescence , Male , Precursor Cell Lymphoblastic Leukemia-Lymphoma/therapy , Treatment Outcome , Young AdultABSTRACT
Switches from the lymphoid to myeloid lineage during B-cell precursor acute lymphoblastic leukemia (BCP-ALL) treatment are considered rare and thus far have been detected in MLL-rearranged leukemia. Here, we describe a novel BCP-ALL subset, switching BCP-ALL or swALL, which demonstrated monocytosis early during treatment. Despite their monocytic phenotype, 'monocytoids' share immunoreceptor gene rearrangements with leukemic B lymphoblasts. All swALLs demonstrated BCP-ALL with CD2 positivity and no MLL alterations, and the proportion of swALLs cases among BCP-ALLs was unexpectedly high (4%). The upregulation of CEBPα and demethylation of the CEBPA gene were significant in blasts at diagnosis, prior to the time when most of the switching occurs. Intermediate stages between CD14(neg)CD19(pos)CD34(pos) B lymphoblasts and CD14(pos)CD19(neg)CD34(neg) 'monocytoids' were detected, and changes in the expression of PAX5, PU1, M-CSFR, GM-CSFR and other genes accompanied the switch. Alterations in the Ikaros and ERG genes were more frequent in swALL patients; however, both were altered in only a minority of swALLs. Moreover, switching could be recapitulated in vitro and in mouse xenografts. Although children with swALL respond slowly to initial therapy, risk-based ALL therapy appears the treatment of choice for swALL. SwALL shows that transdifferentiating into monocytic lineage is specifically associated with CEBPα changes and CD2 expression.
Subject(s)
CD2 Antigens/immunology , Monocytes/pathology , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/immunology , Adolescent , Cell Lineage , Child , Child, Preschool , Cohort Studies , Female , Humans , Immunophenotyping , Male , Multiplex Polymerase Chain Reaction , Neoplasm, Residual , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , PrognosisABSTRACT
Infants <1 year of age have a high prevalence of prognostically unfavorable leukemias and a presumed susceptibility to treatment-related toxicities. A total of 125 infants with acute myeloid leukemia (AML) were treated in studies AML-BFM-98 (n = 59) and -2004 (n = 66). Treatment regimens of both studies were comparable, consisting of intensive induction followed by four courses (mainly high-dose cytarabine and anthracyclines). Allogeneic-hematopoietic stem-cell-transplantation (allo-HSCT) in 1st remission was optional for high-risk (HR) patients. Most infants (120/125=96%) were HR patients according to morphological, cytogenetic/molecular genetic and response criteria. Five-year overall survival was 66 ± 4%, and improved from 61 ± 6% in study-98 to 75 ± 6% in study-2004 (P(logrank) 0.14) and event-free survival rates were 44 ± 6% and 51 ± 6% (P(logrank) 0.66), respectively. Results in HR infants were similar to those of older HR children (1-<2- or 2-<10-year olds, P(logrank) 0.90 for survival). Survival rates of HSCT in 1st remission, initial partial response and after relapse were high (13/14, 2/8 and 20/30 patients, respectively). The latter contributes to excellent 5-year survival after relapse (50±8%). Despite more severe infections and pulmonary toxicities in infants, treatment-related death rate was identical to that of older children (3%). Our data indicate that intensive frontline and relapse AML treatment is feasible in infants, toxicities are manageable, and outcome is favorable.
Subject(s)
Leukemia, Myeloid, Acute/therapy , Anthracyclines/administration & dosage , Child , Child, Preschool , Cytarabine/administration & dosage , Female , Hematopoietic Stem Cell Transplantation , Histone-Lysine N-Methyltransferase , Humans , Infant , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/mortality , Male , Multivariate Analysis , Myeloid-Lymphoid Leukemia Protein/genetics , Salvage Therapy , Treatment OutcomeSubject(s)
Chromosomes, Human, Pair 10/genetics , Chromosomes, Human, Pair 11/genetics , Leukemia, Megakaryoblastic, Acute/genetics , Oncogene Fusion , Oncogene Proteins, Fusion/genetics , Translocation, Genetic , Chromosome Breakage , Chromosomes, Human, Pair 10/ultrastructure , Chromosomes, Human, Pair 11/ultrastructure , Humans , Monomeric Clathrin Assembly Proteins/genetics , RNA, Messenger/genetics , RNA, Neoplasm/genetics , Transcription Factors/geneticsABSTRACT
The optimal therapy for children with relapsed or refractory acute promyelocytic leukemia (APL) is unclear. We therefore reviewed our institutional outcomes for children undergoing allogeneic hematopoietic stem cell transplantation (HSCT) for advanced APL. Between 1986 and 2003, 12 allogeneic HSCTs (five related donor, seven unrelated donor) were performed for 11 patients (median age, 13 years) with relapsed (n = 8) or refractory (n = 3) APL. All patients engrafted, after a median of 18.5 days. Grade B-D acute graft-versus-host disease (GVHD) developed after five transplants (42%; 90% CI, 18-68%), and the cumulative incidence of chronic GVHD was 45% (90% CI, 19-71%). The cumulative incidence of overt relapse post-HSCT was 10% (90% CI, 0-28%). The overall 5-year survival was 73% (90% confidence interval (CI), 51-95%), with a median post-HSCT follow-up of 64 months. The Lansky/Karnofsky performance scores are 100% in six of eight survivors. In view of the low risk of subsequent relapse and favorable survival suggested by other reports and our own experience, we continue to recommend allogeneic HSCT for children with advanced APL for whom a suitably HLA-matched donor is identified.
Subject(s)
Leukemia, Promyelocytic, Acute/therapy , Stem Cell Transplantation , Acute Disease , Adolescent , Adult , Child , Child, Preschool , Chronic Disease , Female , Graft vs Host Disease/epidemiology , Histocompatibility Testing , Humans , Incidence , Male , Recurrence , Retrospective Studies , Stem Cell Transplantation/adverse effects , Survival Analysis , Transplantation, Homologous/adverse effects , Treatment OutcomeABSTRACT
Interphase chromatin is arranged into topologically separated domains comprising gene expression and replication units through genomic sequence elements, so-called MAR or SAR regions (for matrix- or scaffold-associating regions). S/MAR regions are located near the boundaries of actively transcribed genes and were shown to influence their activity. We show that scaffold attachment factor B (SAF-B), which specifically binds to S/MAR regions, interacts with RNA polymerase II (RNA pol II) and a subset of serine-/arginine-rich RNA processing factors (SR proteins). SAF-B localized to the nucleus in a speckled pattern that coincided with the distribution of the SR protein SC35. Furthermore, we show that overexpressed SAF-B induced an increase of the 10S splice product using an E1A reporter gene and repressed the activity of an S/MAR flanked CAT reporter gene construct in vivo . This indicates an association of SAF-B with SR proteins and components of the transcription machinery. Our results describe the coupling of a chromatin organizing S/MAR element with transcription and pre-mRNA processing components and we propose that SAF-B serves as a molecular base to assemble a 'transcriptosome complex' in the vicinity of actively transcribed genes.
Subject(s)
DNA-Binding Proteins/metabolism , Matrix Attachment Region Binding Proteins , Nuclear Matrix-Associated Proteins , Nuclear Proteins/metabolism , RNA Precursors , RNA Splicing , Receptors, Estrogen , Transcription, Genetic , 3T3 Cells , Animals , Binding Sites , Cell Line , Cell Nucleus/metabolism , Chloramphenicol O-Acetyltransferase/genetics , DNA-Binding Proteins/genetics , Genes, Reporter , Humans , Mice , Nuclear Proteins/genetics , Nucleic Acid Hybridization , Phosphoproteins/metabolism , Phosphorylation , RNA Polymerase II/metabolism , RNA-Binding Proteins , Rats , Saccharomyces cerevisiae , Serine-Arginine Splicing FactorsSubject(s)
Fibromuscular Dysplasia/diagnosis , Heart Failure/diagnosis , Hypertrophy, Left Ventricular/diagnosis , Renal Artery Obstruction/diagnosis , Angiography , Angioplasty, Balloon , Echocardiography , Electrocardiography , Fibromuscular Dysplasia/complications , Fibromuscular Dysplasia/therapy , Follow-Up Studies , Heart Failure/complications , Heart Failure/therapy , Humans , Hypertrophy, Left Ventricular/complications , Hypertrophy, Left Ventricular/therapy , Infant , Male , Renal Artery Obstruction/complications , Renal Artery Obstruction/therapyABSTRACT
The largest subunit of the RNA polymerase II (pol II) contains at the carboxy-terminus a peculiar repetitive sequence that consists of 52 tandem repeats of the consensus motif Tyr-Ser-Pro-Thr-Ser-Pro-Ser, referred to as the C-terminal domain (CTD). Upon transcriptional initiation/promoter clearance, the CTD becomes extensively phosphorylated and apparently remains so during elongation. While the underphosphorylated CTD plays a role in transcriptional initiation, recent evidence couples the highly phosphorylated CTD to RNA processing, namely polyadenylation and splicing. Using a yeast two-hybrid screen, we have selected for human proteins that interact with the CTD of RNA polymerase II. The CTD-GAL fusion protein used as a bait is highly phosphorylated in yeast and, accordingly, we did not isolate proteins implicated in transcriptional regulation but rather proteins with possible roles in RNA splicing. One major cDNA clone isolated this way encodes SRrp129/CASP11, a protein that contains a conserved CTD-interaction domain at the C-terminus and an internal serine-arginine rich domain (SR domain). Proteins of the SR family have been implicated in RNA splicing, notably in the regulation of alternative splicing. Thus we consider it likely that SRrp129 is an auxiliary splice factor. We also improved our method to quickly map domains involved in protein-protein interaction (Stagljar et al., 1996, BioTechniques 21, 430-432). Instead of using sonication for the production of a random DNA fragment library, we took advantage of the fact that DNAse I in the presence of manganese (II) produces double strand rather than single strand DNA breaks. The DNA fragment library of the SRrp129 clone was then used in the yeast two-hybrid system to identify the 100-amino acid domain that interacts with the CTD of RNA polymerase II.
Subject(s)
Nuclear Proteins/metabolism , Phosphoproteins/metabolism , RNA Polymerase II/metabolism , Alternative Splicing , Amino Acid Sequence , Humans , Molecular Sequence Data , Nuclear Proteins/genetics , Nuclear Proteins/isolation & purification , Phosphoproteins/genetics , Phosphoproteins/isolation & purification , Protein Structure, Tertiary , RNA Polymerase II/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/isolation & purification , RNA-Binding Proteins/metabolism , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Serine-Arginine Splicing FactorsABSTRACT
The largest subunit of RNA polymerase II shows a striking difference in the degree of phosphorylation, depending on its functional state: initiating and elongating polymerases are unphosphorylated and highly phosphorylated respectively. Phosphorylation mostly occurs at the C-terminal domain (CTD), which consists of a repetitive heptapeptide structure. Using the yeast two-hybrid system, we have selected for mammalian proteins that interact with the phosphorylated CTD of mammalian RNA polymerase II. A prominent isolate, designated SRcyp/CASP10, specifically interacts with the CTD not only in vivo but also in vitro . It contains a serine/arginine-rich (SR) domain, similar to that found in the SR protein family of pre-mRNA splicing factors, which is required for interaction with the CTD. Most remarkably, the N-terminal region of SRcyp includes a peptidyl-prolyl cis - trans isomerase domain characteristic of immunophilins/cyclophilins (Cyp), a protein family implicated in protein folding, assembly and transport. SRcyp is a nuclear protein with a characteristic distribution in large irregularly shaped nuclear speckles and co-localizes perfectly with the SR domain-containing splicing factor SC35. Recent independent investigations have provided complementary data, such as an association of the phosphorylated form of RNA polymerase II with the nuclear speckles, impaired splicing in a CTD deletion background and inhibition of in vitro splicing by CTD peptides. Taken together, these data indicate that factors directly or indirectly involved in splicing are associated with the elongating RNA polymerases, from where they might translocate to the nascent transcripts to ensure efficient splicing, concomitant with transcription.