Prognostic impact of CEBPA bZIP domain mutation in acute myeloid leukemia

Mutations of CCAAT/enhancer binding protein alpha (CEBPAmu) are found in 10-15% of de novo acute myeloid leukemia (AML) cases. Double-mutated CEBPA (CEBPAdm) is associated with a favorable prognosis; however, single-mutated CEBPA (CEBPAsm) does not appear to improve prognosis. We investigated the CEBPAmu for prognosis in 1028 AML patients, registered in the Multi-center Collaborative Program for Gene Sequencing of Japanese AML. It was found that CEBPAmu in the basic leucine zipper domain (bZIP) was strongly associated with a favorable prognosis, but CEBPAmu out of the bZIP domain was not. The presence of CEBPAmu in bZIP was a strong indicator of a higher chance of achieving complete remission (p<0.001), better overall survival (OS; p<0.001) and a lower risk of relapse (p<0.001). The prognostic significance of CEBPAmu in bZIP was also observed in the subgroup with CEBPAsm (all patients, OS: p=0.008; the cumulative incidence of relapse (CIR): p=0.063. patients aged ≤70 years and with intermediate-risk karyotype, OS: p=0.008; CIR: p=0.026). Multivariate analysis of 744 patients aged ≤70 years showed that CEBPAmu in bZIP was the most potent predictor of OS (hazard ratio: 0.3287; p<0.001). CEBPAdm was validated as a cofounding factor, which was overlapping with CEBPAmu in bZIP. In summary, these findings indicate that CEBPAmu in bZIP is a potent marker for AML prognosis. It holds potential in the refinement of treatment stratification and the development of targeted therapeutic approaches in CEBPA mutated AML.

The transcription factor Xrp1 orchestrates both reduced translation and cell competition upon defective ribosome assembly or function

Ribosomal Protein (Rp) gene haploinsufficiency affects overall translation rate, leads to cell elimination by competition with wild type cells in mosaic tissues, and sometimes leads to accumulation of protein aggregates. The changes in ribosomal subunit levels observed are not sufficient for these effects, which all depend on the AT-hook, bZip domain protein Xrp1. In Rp+/- cells, Xrp1 reduced global translation through PERK-dependent phosphorylation of eIF2α. eIF2α phosphorylation was sufficient to reduce translation in, and also enable cell competition of, otherwise wild type cells. Unexpectedly, however, many other defects reducing ribosome biogenesis or function (depletion of TAF1B, eIF2, eIF4G, eIF6, eEF2, eEF1α1, or eIF5A), also increased eIF2α phosphorylation and enabled cell competition. In all cases this was through the Xrp1 expression that was induced, placing Xrp1 as the downstream instigator of cell competition that also contributed to overall translation deficits. In the absence of Xrp1, translation differences between cells were not themselves sufficient to trigger cell competition. Thus, Xrp1, which is shown here to be a sequence-specific transcription factor, is the master regulator that triggers cell competition and other consequences of multiple ribosomal stresses.

Transmission of light signals from the light-oxygen-voltage core via the hydrophobic region of the β-sheet surface in aureochrome-1

Light-Oxygen-Voltage (LOV) domains are responsible for detecting blue light (BL) and regulating the activities of effector domains in various organisms. Photozipper (PZ), an N-terminally truncated aureochrome-1 protein, contains a LOV domain and a basic leucin zipper (bZIP) domain and plays a role as a light-activatable transcription factor. PZ is monomeric in the dark state and undergoes non-covalent dimerization upon illumination with BL, subsequently increasing its affinity for the target DNA. To clarify the molecular mechanism of aureochromes, we prepared site-directed mutants of PZ and performed quantitative analyses in the dark and light states. Although the amino acid substitutions in the hinge region between the LOV core and A’α helix had minor effects on the dimerization and DNA-binding properties of PZ, the substitutions in the β-sheet region of the LOV core and in the A’α helix significantly affected these properties. We found that light signals are transmitted from the LOV core to the effector bZIP domain via the hydrophobic residues on the β-sheet. The light-induced conformational change possibly deforms the hydrophobic regions of the LOV core and induces the detachment of the A’α helix to expose the dimerization surface, likely activating the bZIP domain in a light-dependent manner.

BACH1 recruits NANOG and histone H3 lysine 4 methyltransferase MLL/SET1 complexes to regulate enhancer–promoter activity and maintains pluripotency

Maintenance of stem-cell identity requires proper regulation of enhancer activity. Both transcription factors OCT4/SOX2/NANOG and histone methyltransferase complexes MLL/SET1 were shown to regulate enhancer activity, but how they are regulated in embryonic stem cells (ESCs) remains further studies. Here, we report a transcription factor BACH1, which directly interacts with OCT4/SOX2/NANOG (OSN) and MLL/SET1 methyltransferase complexes and maintains pluripotency in mouse ESCs (mESCs). BTB domain and bZIP domain of BACH1 are required for these interactions and pluripotency maintenance. Loss of BACH1 reduced the interaction between NANOG and MLL1/SET1 complexes, and decreased their occupancy on chromatin, and further decreased H3 lysine 4 trimethylation (H3K4me3) level on gene promoters and (super-) enhancers, leading to decreased enhancer activity and transcription activity, especially on stemness-related genes. Moreover, BACH1 recruited NANOG through chromatin looping and regulated remote NANOG binding, fine-tuning enhancer–promoter activity and gene expression. Collectively, these observations suggest that BACH1 maintains pluripotency in ESCs by recruiting NANOG and MLL/SET1 complexes to chromatin and maintaining the trimethylated state of H3K4 and enhancer–promoter activity, especially on stemness-related genes.

Genome-Wide Identification and Expression Analysis of the bZIP Transcription Factors in the Mycoparasite Coniothyrium minitans

The basic leucine zipper (bZIP) proteins family is one of the largest and most diverse transcription factors, widely distributed in eukaryotes. However, no information is available regarding the bZIP gene family in Coniothyrium minitans, an important biocontrol agent of the plant pathogen Sclerotinia sclerotiorum. In this study, we identified 34 bZIP genes from the C. minitans genome, which were classified into 8 groups based on their phylogenetic relationships. Intron analysis showed that 28 CmbZIP genes harbored a variable number of introns, and 15 of them shared a feature that intron inserted into the bZIP domain. The intron position in bZIP domain was highly conserved, which was related to recognize the arginine (R) and could be treated as a genomic imprinting. Expression analysis of the CmbZIP genes in response to abiotic stresses indicated that they might play distinct roles in abiotic stress responses. Results showed that 22 CmbZIP genes were upregulated during the later stage of conidial development. Furthermore, transcriptome analysis indicated that CmbZIP genes are involved in different stages of mycoparasitism. Among deletion mutants of four CmbZIPs (CmbZIP07, -09, -13, and -16), only ΔCmbZIP16 mutants significantly reduced its tolerance to the oxidative stress. The other mutants exhibited no significant effects on colony morphology, mycelial growth, conidiation, and mycoparasitism. Taken together, our results suggested that CmbZIP genes play important roles in the abiotic stress responses, conidial development, and mycoparasitism. These results provide comprehensive information of the CmbZIP gene family and lay the foundation for further research on the bZIP gene family regarding their biological functions and evolutionary history.

Somatic Bzip Mutations of CEBPA Are Associated with Favorable Outcome Regardless of Presence As Single Vs. Double Mutation

Somatic mutations of CEBPA in AML are present at similar rates in all age groups and have been shown to be associated with favorable outcome. The most common cooperating variant in CEBPA mutations is a second CEBPA mutation, where the cooperation of TAD and bZip mutations lead to a highly penetrant variant which is sufficient for malignant transformation. Data suggested that only double CEBPA mutations (CEBPA-dm) were associated with favorable outcome. Currently, WHO classification of AML includes CEBPA-dm as a diagnostic entity in AML. We previously reported that in children and young adults, somatic single CEBPA mutations, which almost exclusively occurred in the bZip domain, had similar outcomes to CEBPA-dm patients. We have also shown that CSF3R and GATA2 mutations occur in childhood AML and are highly associated with CEBPA mutations. In this large analysis, we sought to define the prognostic impact of CEBPA-dm vs single bZip mutations and evaluate the impact of common co-occurring mutations. This study included a total of 2,958 patients (age 0-29.94 years) treated on the 4 large Children's Oncology Group trials CCG2961 (n=562), AAML03P1 (n=266), AAML0531 (n=917), and AAML1031(n=1233). DNA binding domain of CEBPA (bZip domain) was analyzed by fragment length analysis and 160 patients with mutations were identified and verified with sanger sequencing (n=23 2961; n=15 03P1; n=51 0531; n=71 1031). In all cases where bZip mutations were detected, N terminal region was sequenced for identification of TAD mutations. Of the 160 cases with bZip mutation, 132 patients had a secondary TAD mutation (CEBPA-dm; 82.5%) and in the remaining 28 cases (17.5%) no TAD mutation was detected (CEBPA-sm). Comprehensive NGS data was available in a cohort of patients (n=106) and demonstrated dm vs. sm prevalence of 81% and 19% respectively, similar to the cohort overall. We compared clinical and biologic characteristics among CEBPA-dm vs. CEBPA-sm patients. The two groups showed no significant differences across karyotype, complete remission rates, FLT3/ITD or NPM1 status, cyto-molecular risk group, age, or diagnostic WBC. CEBPA mutations were more prevalent in Asian patients (10.5%) compared to other races (p=0.019). Evaluation of outcomes for those with CEBPA-dm vs. sm in the entire cohort demonstrated that those with CEBPA bZip mutations regardless of dm vs. sm status had identical event free survival (EFS) of 64% (Fig 1A; p=0.777). Similarly, the overall survival (OS) for CEBPA-sm was 89% vs. 81% for CEBPA-dm (p=0.259; Figure 1A). Evaluation by study cohort demonstrated that presence of bZip mutations whether as sm or dm had similar favorable clinical impact (Figure 1B-D; p=NS in each study for EFS and OS). Analysis of CEBPA+ patients with comprehensive NGS data identified somatic mutations in GATA2 (n=20; 19%) and CSF3R (n=12; 11%)(Figure 1E). Co-occurrence of a GATA2 mutation did not impact the favorable outcome conferred by CEBPA+ on EFS or OS (p=0.356 and p=0.749 respectively; Figure 1F). However, presence of CSF3R mutation significantly modulated the clinical implications of CEBPA mutations. We previously showed an overlap with CEBPA and CSF3R mutations, which occur as SNVs in the extracellular domain or truncating mutations in the cytoplasmic domain. CSF3R mutations result in aberrant CSF3R activation and expression and have been shown to be amenable to targeted therapy with either JAK or SRC family inhibitors. Given the large number of CEBPA+ patients available for analysis, we evaluated the outcomes of patients with CEBPA+/CSF3R+ (n=12) and without CSF3R mutations (n=94). Dual mutant CEBPA+/CSF3R+ patients had a significantly inferior EFS of 17% vs. 63% (p<0.001; Figure 1F) due to high rate of relapse. Importantly, despite high relapse rates these dual mutant patients were salvageable after relapse with an OS of 73% vs. 83% for CEBPA+ patients without CSF3R (p=0.563). In a large study of CEBPA mutations in pediatric AML, we show that patients with a bZip mutation, regardless of sm vs. dm status have a favorable prognosis. Further, we confirm the significant overlap of CEBPA and CSF3R mutations previously reported, and demonstrate that CEBPA+/CSF3R+ patients are at high risk for relapse and thus should not be considered a low risk cohort. Given the poor outcomes with current regimens, patients with CEBPA+/CSF3R+ mutations could be considered for addition of tyrosine kinase inhibitors with upfront therapy. Disclosures No relevant conflicts of interest to declare.

A Noncanonical Basic Motif of Epstein-Barr Virus ZEBRA Protein Facilitates Recognition of Methylated DNA, High-Affinity DNA Binding, and Lytic Activation

ABSTRACTThe pathogenesis of Epstein-Barr virus (EBV) infection, including development of lymphomas and carcinomas, is dependent on the ability of the virus to transit from latency to the lytic phase. This conversion, and ultimately disease development, depends on the molecular switch protein, ZEBRA, a viral bZIP transcription factor that initiates transcription from promoters of viral lytic genes. By binding to the origin of viral replication, ZEBRA is also an essential replication protein. Here, we identified a novel DNA-binding motif of ZEBRA, N terminal to the canonical bZIP domain. This RRTRK motif is important for high-affinity binding to DNA and is essential for recognizing the methylation state of viral promoters. Mutations in this motif lead to deficiencies in DNA binding, recognition of DNA methylation, lytic cycle DNA replication, and viral late gene expression. This work advances our understanding of ZEBRA-dependent activation of the viral lytic cascade.IMPORTANCEThe binding of ZEBRA to methylated and unmethylated viral DNA triggers activation of the EBV lytic cycle, leading to viral replication and, in some patients, cancer development. Our work thoroughly examines how ZEBRA uses a previously unrecognized basic motif to bind nonmethylated and methylated DNA targets, leading to viral lytic activation. Our findings show that two different positively charged motifs, including the canonical BZIP domain and a newly identified RRTRK motif, contribute to the mechanism of DNA recognition by a viral AP-1 protein. This work contributes to the assessment of ZEBRA as a potential therapeutic target for antiviral and oncolytic treatments.

Transcriptional regulatory activity of the cereal grain bZip protein TaABF1 can be either stimulated or inhibited by phosphorylation

The wheat bZip transcription factor TaABF1 mediates both abscisic acid (ABA)-induced and ABA-suppressed gene expression. As levels of TaABF1 protein do not change in response to ABA, and TaABF1 is in a phosphorylated state in vivo, we investigated whether TaABF1 could be regulated at the post-translational level. In bombarded aleurone cells, a TaABF1 protein carrying phosphomimetic mutations (serine to aspartate) at four sites (S36D, S37D, S113D, S115D) was three to five times more potent than wild-type TaABF1 in activating HVA1, an ABA-responsive gene. The phosphomimetic mutations also increased the ability of TaABF1 to downregulate the ABA-suppressed gene Amy32b. These findings strongly suggest that phosphorylation at these sites increases the transcriptional regulatory activity of TaABF1. In contrast to the activation observed by the quadruple serine to aspartate mutation, a single S113D mutation completely eliminated the ability of TaABF1 to upregulate HVA1 or downregulate Amy32b. Thus phosphorylation of TaABF1 can either stimulate or inhibit the activity of TaABF1 in regulating downstream genes, depending on the site and pattern of phosphorylation. Mutation of S318 and S322 (in the bZIP domain) eliminated the ability of TaABF1 to activate HVA1, but had no effect on the ability of TaABF1 to downregulate Amy32b, suggesting that TaABF1 represses Amy32b expression through a mechanism other than direct DNA binding. An important step towards understanding how ABA and gibberellin (GA) signals are integrated through TaABF1 phosphorylation to regulate downstream gene expression is to clarify the effects of those hormones on the expression of specific genes. In contrast to some other ABA-induced genes, we found that HVA1 induction by ABA or TaABF1 is not inhibited by GA.