B-myb Gene Research Articles

G-quadruplex forming motifs in the promoter region of the B-MYB proto-oncogene

To combat cancer, a comprehensive understanding of the molecular mechanisms and behaviors involved in carcinogenesis is crucial, as tumorigenesis is a complex process influenced by various genetic events and disease hallmarks. The B-MYB gene encodes a transcription factor involved in cell cycle regulation, survival, and differentiation in normal cells. B-MYB can be transformed into an oncogene through mutations, and abnormal expression of B-MYB has been identified in various cancers, including lung cancer, and is associated with poor prognosis. Targeting this oncogene is a promising approach for anti-cancer drug design. B-MYB has been deemed undruggable in previous reports, necessitating the search for novel therapeutic options. In this study, we found that the B-MYB gene promoter contains several G/C rich motifs compatible with G-quadruplex (G4) formation. We investigated and validated the existence of G4 structures in the promoter region of B-MYB, first in vitro using a combination of bioinformatics, biophysical, and biochemical methods, then in cell with the recently developed G4access method.

Targeting proto-oncogene B-MYB G-quadruplex with a nucleic acid–based fluorescent probe

The B-MYB gene encodes a transcription factor (B-MYB) that regulates cell growth and survival. Abnormal expression of B-MYB is frequently observed in lung cancer and poses challenges for targeted drug therapy. Oncogenes often contain DNA structures called G-quadruplexes (G4s) in their promoter regions, and B-MYB is no exception. These G4s play roles in genetic regulation and are potential cancer treatment targets.In this study, a probe was designed to specifically identify a G4 within the promoter region of the B-MYB gene. This probe combines an acridine derivative ligand with a DNA segment complementary to the target sequence, enabling it to hybridize with the adjacent sequence of the G4 being investigated.Biophysical studies demonstrated that the acridine derivative ligands C5NH2 and C8NH2 not only effectively stabilized the G4 structure but also exhibited moderate affinity. They were capable of altering the G4 topology and exhibited enhanced fluorescence emission in the presence of this quadruplex. Additionally, these ligands increased the number of G4s observed in cellular studies. Through various biophysical studies, the target sequence was shown to form a G4 structure, even with an extra nucleotide tail added to its flanking region. Cellular studies confirmed the co-localization between the target sequence and the developed probe.

Oncogenic B-Myb Is Associated With Deregulation of the DREAM-Mediated Cell Cycle Gene Expression Program in High Grade Serous Ovarian Carcinoma Clinical Tumor Samples.

Cell cycle control drives cancer progression and treatment response in high grade serous ovarian carcinoma (HGSOC). MYBL2 (encoding B-Myb), an oncogene with prognostic significance in several cancers, is highly expressed in most HGSOC cases; however, the clinical significance of B-Myb in this disease has not been well-characterized. B-Myb is associated with cell proliferation through formation of the MMB (Myb and MuvB core) protein complex required for transcription of mitotic genes. High B-Myb expression disrupts the formation of another transcriptional cell cycle regulatory complex involving the MuvB core, DREAM (DP, RB-like, E2F, and MuvB), in human cell lines. DREAM coordinates cell cycle dependent gene expression by repressing over 800 cell cycle genes in G0/G1. Here, we take a bioinformatics approach to further evaluate the effect of B-Myb expression on DREAM target genes in HGSOC and validate our cellular model with clinical specimens. We show that MYBL2 is highly expressed in HGSOC and correlates with expression of DREAM and MMB target genes in both The Cancer Genome Atlas (TCGA) as well as independent analyses of HGSOC primary tumors (N = 52). High B-Myb expression was also associated with poor overall survival in the TCGA cohort and analysis by a DREAM target gene expression signature yielded a negative impact on survival. Together, our data support the conclusion that high expression of MYBL2 is associated with deregulation of DREAM/MMB-mediated cell cycle gene expression programs in HGSOC and may serve as a prognostic factor independent of its cell cycle role. This provides rationale for further, larger scale studies aimed to determine the clinical predictive value of the B-Myb gene expression signature for treatment response as well as patient outcomes.

Antibiosis and bmyB Gene Presence As Prevalent Traits for the Selection of Efficient Bacillus Biocontrol Agents against Crown Gall Disease.

This study aimed to improve the screening method for the selection of Bacillus biocontrol agents against crown gall disease. The relationship between the strain biocontrol ability and their in vitro studied traits was investigated to identify the most important factors to be considered for the selection of effective biocontrol agents. In fact, previous selection procedure relying only on in vitro antibacterial activity was shown to be not suitable in some cases. A direct plant-protection strategy was performed to screen the 32 Bacillus biocontrol agent candidates. Moreover, potential in vitro biocontrol traits were investigated including biofilm formation, motility, hemolytic activity, detection of lipopeptide biosynthetic genes (sfp, ituC and bmyB) and production of antibacterial compounds. The obtained results indicated high correlations of the efficiency of the biocontrol with the reduction of gall weight (p = 0.000) and the antibacterial activity in vitro (p = 0.000). Moreover, there was strong correlations of the efficiency of the biocontrol (p = 0.004) and the reduction in gall weight (p = 0.000) with the presence of the bmyB gene. This gene directs the synthesis of the lipopeptide bacillomycin belonging to the iturinic family of lipopeptides. These results were also confirmed by the two-way hierarchical cluster analysis and the correspondence analysis showing the relatedness of these four variables. According to the obtained results a new screening procedure of Bacillus biocontrol agents against crown gall disease could be advanced consisting on two step selection procedure. The first consists on selecting strains with high antibacterial activity in vitro or those harbouring the bmyB gene. Further selection has to be performed on tomato plants in vivo. Moreover, based on the results of the biocontrol assay, five potent strains exhibiting high biocontrol abilities were selected. They were identified as Bacillus subtilis or Bacillus amyloliquefaciens. These strains were found to produce either surfactin or surfactin and iturin lipopeptides. In conclusion, our study presented a new and effective method to evaluate the biocontrol ability of antagonistic Bacillus strains against crown gall disease that could increase the efficiency of screening method of biocontrol agents. Besides, the selected strains could be used as novel biocontrol agents against pathogenic Agrobacterium tumefaciens strains.

B-Myb, Cancer, Senescence, and MicroRNAs

The transcription factor B-Myb plays a critical role in regulating gene expression and is implicated in controlling carcinogenesis and cellular senescence. Transcription of the B-Myb gene is regulated by retinoblastoma proteins acting directly on the B-Myb promoter. Recently, we found that microRNAs also control the abundance of B-Myb mRNA during senescence, adding another level of complexity to B-Myb regulation. This review focuses on the importance of B-Myb in cancer and senescence, with an emphasis on the regulation of B-Myb expression and activity.

Activation of v-Myb avian myeloblastosis viral oncogene homolog-like2 (MYBL2)-LIN9 complex contributes to human hepatocarcinogenesis and identifies a subset of hepatocellular carcinoma with mutant p53

Up-regulation of the v-Myb avian myeloblastosis viral oncogene homolog-like2 B-Myb (MYBL2) gene occurs in human hepatocellular carcinoma (HCC) and is associated with faster progression of rodent hepatocarcinogenesis. We evaluated, in distinct human HCC prognostic subtypes (as defined by patient survival length), activation of MYBL2 and MYBL2-related genes, and relationships of p53 status with MYBL2 activity. Highest total and phosphorylated protein levels of MYBL2, E2F1-DP1, inactivated retinoblastoma protein (pRB), and cyclin B1 occurred in HCC with poorer outcome (HCCP), compared to HCC with better outcome (HCCB). In HCCP, highest LIN9-MYBL2 complex (LINC) and lowest inactive LIN9-p130 complex levels occurred. MYBL2 positively correlated with HCC genomic instability, proliferation, and microvessel density, and negatively with apoptosis. Higher MYBL2/LINC activation in HCC with mutated p53 was in contrast with LINC inactivation in HCC harboring wildtype p53. Small interfering RNA (siRNA)-mediated MYBL2/LINC silencing reduced proliferation, induced apoptosis, and DNA damage at similar levels in HCC cell lines, irrespective of p53 status. However, association of MYBL2/LINC silencing with doxorubicin-induced DNA damage caused stronger growth restraint in p53(-/-) Huh7 and Hep3B cells than in p53(+/+) Huh6 and HepG2 cells. Doxorubicin triggered LIN9 dissociation from MYBL2 in p53(+/+) cell lines and increased MYBL2-LIN9 complexes in p53(-/-) cells. Doxorubicin-induced MYBL2 dissociation from LIN9 led to p21(WAF1) up-regulation in p53(+/+) but not in p53(-/-) cell lines. Suppression of p53 or p21(WAF1) genes abolished DNA damage response, enhanced apoptosis, and inhibited growth in doxorubicin-treated cells harboring p53(+/+) . We show that MYBL2 activation is crucial for human HCC progression. In particular, our data indicate that MYBL2-LIN9 complex integrity contributes to survival of DNA damaged p53(-/-) cells. Thus, MYBL2 inhibition could represent a valuable adjuvant for treatments against human HCC with mutated p53.

Addiction to B-MYB.

Neuroblastoma, the most common and deadly extracranial solid tumor of childhood, has long provided a challenge to both clinicians and basic scientists. Despite its high prevalence among pediatric cancers and extensive efforts over the last several decades, there is still been only marginal improvement in overall survival and almost no improvement in survival for high-risk neuroblastoma [1]. Numerous risk stratification studies have shown that amplification of the MYCN oncogene is the clearest clinical indicator of poor prognosis. Despite the importance of MYCN amplification in neuroblastoma, little work has been done to examine the transcriptional regulation of the MYCN amplicon. In addition to transcriptional regulation, a second primary means through which cells regulate levels of Mycn is at the level of protein stability. Therefore, exploring methods enabling targeting of Mycn protein stability is a major focus of current therapeutic research (reviewed in [2]). If significant transcriptional regulation of the MYCN amplicon in neuroblastoma could be characterized and exploited, these could result in novel strategies to target this deadly disease. What do we know about transcriptional regulation of MYCN in neuroblastoma? In conjunction with binding partners including Max and Miz, Myc family transcription factors (including c-MYC, MYCN, and MYCL) bind to specific “E-box” elements to regulate gene expression. E-boxes are quite common in the genome and are present in the promoter regions of approximately 25% of known genes indicating complex and tissue specific regulation of Myc mediated transcription [3]. While many downstream transcriptional targets of c-Myc have been determined (including the B-MYB gene)[4], detailed analysis and confirmation of MYCN regulated genes is of vital importance to our understanding of MYCN mediated oncogenesis. In this issue of Oncotarget, Gualdrini et al propose the B-MYB transcription factor as one such target, and provide preliminary evidence for a reciprocal regulation between the amplified MYCN and the B-MYB transcription factors [5]. Expression of the B-MYB transcription factor has been previously shown to be a poor prognostic indicator in neuroblastoma, independent of MYCN amplification [6]. In this study, Gualdrini et al show an intriguing correlation between expression of mRNA for MYCN and B-MYB in neuroblastoma patient samples amplified for MYCN, extending this observation to the protein level in several MYCN amplified neuroblastoma cell lines. Importantly, the correlation between MYCN and B-MYB does not hold true in MYCN non-amplified samples. Additionally, using CHIP in established cell lines, the authors provide preliminary evidence that Mycn protein can bind to and induce transcription of B-MYC, consistent with B-MYB as a transcriptional target of MYCN. Perhaps most importantly, the study shows that knockdown of B-MYB by shRNA blocks proliferation in neuroblastoma cell lines with amplified MYCN. However, mechanistic details of this synthetic anti-proliferative effect between knockdown of B-MYB and amplification of MYCN remain to be determined. Also importantly, the authors use a single shRNA against BMYB, and do not extend this observation to primary tumors, thus limiting the impact of this result. Lastly, B-MYB was also shown to bind to the MYCN promoter in chromatin-IP assays of MYCN amplified neuroblastoma cells in culture, and induced a relatively modest transcriptional upregulation of a MYCN promoter element. The authors use these exciting albeit preliminary results to propose a novel model for a reciprocal regulatory loop between these two transcription factors. If significant epigenetic and transcriptional regulation of amplified MYCN (implied by these studies) can be validated in primary tumors, these studies would support BMYB as target for therapeutic intervention in MYCN amplified cancers. However, given myriad difficulties currently in-place in targeting Mycn itself, the ability to target both BMYC and MYCN in neuroblastoma raises its own group of challenges.

Addiction of MYCN amplified tumours to B-MYB underscores a reciprocal regulatory loop.

MYCN is a member of the MYC family of oncoproteins frequently amplified or overexpressed in aggressive, paediatric tumours of the nervous system. In this study we have identified the gene B-MYB, encoding the transcription factor also known as MYBL2, as a downstream target of MYCN. Using multiple in silico databases we show that expression of B-MYB significantly correlates with that of MYCN in neuroblastoma patients. MYCN binds to and activates the B-MYB gene in vivo and in vitro. Blunting B-MYB expression by RNA interference causes reduced proliferation of MYCN amplified, but not MYCN-non amplified, neuroblastoma cell lines, indicating that tumour cells are addicted to B-MYB in a MYCN dependent manner. Notably, B-MYB binds in vivo to the MYCN amplicon and is required for its expression. We conclude that MYCN and B-MYB are engaged in a reciprocal regulatory loop whose pharmacological targeting could be beneficial to patients with the aggressive forms of cancer in which MYCN is amplified.

Addiction of MYCN amplified tumours to B-MYB underscores a reciprocal regulatory loop.

MYCN is a member of the MYC family of oncoproteins frequently amplified or overexpressed in aggressive, paediatric tumours of the nervous system. In this study we have identified the gene B-MYB, encoding the transcription factor also known as MYBL2, as a downstream target of MYCN. Using multiple in silico databases we show that expression of B-MYB significantly correlates with that of MYCN in neuroblastoma patients. MYCN binds to and activates the B-MYB gene in vivo and in vitro. Blunting B-MYB expression by RNA interference causes reduced proliferation of MYCN amplified, but not MYCN-non amplified, neuroblastoma cell lines, indicating that tumour cells are addicted to B-MYB in a MYCN dependent manner. Notably, B-MYB binds in vivo to the MYCN amplicon and is required for its expression. We conclude that MYCN and B-MYB are engaged in a reciprocal regulatory loop whose pharmacological targeting could be beneficial to patients with the aggressive forms of cancer in which MYCN is amplified.

Isolation and functional assessment of common, polymorphic variants of the B-MYB proto-oncogene associated with a reduced cancer risk

The B-MYB proto-oncogene is a transcription factor belonging to the MYB family that is frequently overexpressed or amplified in different types of human malignancies. While it is suspected that B-MYB plays a role in human cancer, there is still no direct evidence of its causative role. Looking for mutations of the B-MYB gene in human cell lines and primary cancer samples, we frequently isolated two nonsynonymous B-MYB polymorphic variants (rs2070235 and rs11556379). Compared to the wild-type protein, the B-MYB isoforms display altered conformation, impaired regulation of target genes and decreased antiapoptotic activity, suggesting that they are hypomorphic variants of the major allele. Importantly, the B-MYB polymorphisms are common; rs2070235 and rs11556379 are found, depending on the ethnic background, in 10-50% of human subjects. We postulated that, if B-MYB activity is important for transformation, the presence of common, hypomorphic variants might modify cancer risk. Indeed, the B-MYB polymorphisms are underrepresented in 419 cancer patients compared to 230 controls (odds ratio 0.53; (95%) confidence interval 0.385-0.755; P=0.001). This data imply that a large fraction of the human population is carrier of B-MYB alleles that might be associated with a reduced risk of developing neoplastic disease.

Co‐regulation of B‐Myb expression by E2F1 and EGF receptor

Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is frequently over-expressed in human cancers and is associated with tumorigenesis, and increased tumor proliferation and progression. Also found in breast tumors with high levels is B-Myb, a transcription factor whose expression is activated by E2F1/3 at the late G1 phase and the level is sustained through the S phase. Recent reports suggest a casual correlation between EGFR and B-Myb expression in primary breast carcinomas. However, the mechanism for such co-expression remains un-investigated. Here, we report that EGFR is important for B-Myb expression and the underlying mechanism involves cooperated effects from EGFR and E2F1. EGF stimulation and forced expression of EGFR significantly increase B-Myb gene activity and such increase occurs in the G1 phase. EGF-induced B-Myb expression was not significantly suppressed following inhibition of PI-3K and ERK, two major EGFR downstream pathways. In contrast, we observed EGF-induced in vivo association of nuclear EGFR to the B-Myb promoter and the association is only detected at the G1/S phase and is abolished by EGFR kinase inhibitor. As EGFR lacks DNA-binding domain but contains transactivational activity and E2F1 activates B-Myb expression in the G1/S phase, we further reasoned that nuclear EGFR might cooperate with E2F1 leading to activation of B-Myb. Indeed, we found that EGFR co-immunoprecipitated with E2F1 in an EGF-dependent manner and that EGF activated in vivo binding of E2F1 to the B-Myb promoter. Consistently, forced expression of both EGFR and E2F1 in EGFR-null CHO cells greatly enhanced B-Myb promoter activity, compared to the vector control and expression of EGFR or E2F1 alone. Promoter mutagenesis studies showed that EGF-induced activation of B-Myb promoter required both E2F and EGFR target sites. In summary, our data suggest that deregulated EGFR signaling pathway facilitate tumor cell proliferation partly via EGFR interaction with E2F1 and subsequent activation of B-Myb gene expression.

Temperature-dependent Modification and Activation of B-MYB: IMPLICATIONS FOR CELL SURVIVAL

B-MYB is a ubiquitous transcription factor with an essential role in mouse development. Because cells with a disrupted B-MYB gene cannot be obtained, it is still unknown what is the critical function(s) exerted by B-MYB in mammalian cells. In this study we have observed that reducing B-MYB expression in primary human fibroblasts by using RNA interference results in a partial block of the cells in the G(2) phase of the cell cycle and cell death. Surprisingly, suppressing B-MYB transcriptional activity with a dominant-negative molecule is without effect, suggesting that its transactivating function is not essential. Only human or murine fibroblasts exposed to high temperature are sensitized to cell death in the presence of dominant-negative B-MYB. This correlates with temperature-dependent binding of endogenous B-MYB to transcriptional regulatory elements of the stress-related gene ApoJ/clusterin. We find that regulation of ApoJ/clusterin by B-MYB is a pro-survival response to thermal stress. Thus, B-MYB is regulated by temperature to activate genes required for cell survival.

B-Myb Represses Elastin Gene Expression in Aortic Smooth Muscle Cells

B-Myb represses collagen gene transcription in vascular smooth muscle cells (SMCs) in vitro and in vivo. Here we sought to determine whether elastin is similarly repressed by B-Myb. Levels of tropoelastin mRNA and protein were lower in aortas and isolated SMCs of adult transgenic mice expressing the human B-myb gene, driven by the basal cytomegalovirus promoter, compared with age-matched wild type (WT) animals. However, the vessel wall architecture and levels of insoluble elastin revealed no differences. Since elastin deposition occurs early in development, microarray analysis was performed using nontransgenic mice. Aortic levels of tropoelastin mRNA were low during embryonal growth and increased substantially in neonates, whereas B-myb levels varied inversely. Tropoelastin mRNA expression in aortas of 6-day-old neonatal transgenic and WT animals was comparable. Recently, we demonstrated that cyclin A-Cdk2 prevents B-Myb-mediated repression of collagen promoter activity. Cyclin A2 levels were higher in neonatal versus adult WT or transgenic mouse aortas. Ectopic cyclin A expression reversed the ability of B-Myb to repress elastin gene promoter activity in adult SMCs. These results demonstrate for the first time that B-Myb represses SMC elastin gene expression and that cyclin A plays a role in the developmental regulation of elastin gene expression in the aorta. Furthermore, the findings provide additional insight into the mechanism of B-myb-mediated resistance to femoral artery injury.

The Transcription Factor B-Myb Is a Key Regulator of S Phase during Endoreplication in Megakaryocytes.

During their differentiation, and uniquely within haemopoietic cells, megakaryocytes acquire a polyploid nuclear DNA content, most likely to enhance their ability to produce large numbers of platelets for subsequent release into the blood. Polyploidy is achieved through the process of endoreplication, that is, successive S-phases without concomitant mitosis. Although a number of proteins involved in the control of the cell cycle have been implicated in megakaryocytic endoreplication little is known concerning which transcriptional regulators might influence the process. Several transcription factors have been shown to regulate the expression of characteristic functional proteins during megakaryocytic differentiation, but as yet none has been linked to endoreplication. In fact, work with certain model systems for megakarocyte differentiation suggests that the regulation of megakaryocyte-specific genes might be quite separate from the control of endoreplication.From studies on many cell types it is evident that the cell cycle regulated Myb family transcription factor B-Myb is crucial during S-phase. B-Myb expression has been described to be maximal at G1/S phase and it is tightly regulated at the level of both RNA and protein. We have examined the expression and function of B-Myb in both model cell systems for megakaryocyte differentiation and in primary murine cells. It has been described that B-Myb activity needs to be down regulated during terminal differentiation of most haemopoietic cells. However we have found that during differentiation of human megakaryoblastic cell lines induced with phorbol esters or in differentiating primary mouse megakaryocytes, B-myb RNA and protein levels remain constant. To assess the function of B-Myb in megakaryocytic cells we manipulated its level of expression. Over expression of B-Myb in the HEL megakaryoblastic cell line resulted in an increase in the number of cells entering into S-phase. When the HEL cells over expressing B-Myb were treated with phorbol ester to induce differentiation we observed an increase in the fraction of cells actively endoreplicating. On the other hand, reducing B-Myb levels through the use of siRNAs resulted in an accumulation of cells at the G1/S boundary, and through incorporation of BrdU we were able to show that this was due to decreased progression through S-phase. When similar experiments to reduce B-Myb levels were performed on HEL cells undergoing differentiation we observed the same decline in S-phase progression during endoreplicative DNA synthesis. In conclusion, our experiments are the first to identify a key transcriptional regulator of the specialised cell cycle in differentiating megakaryocytes.Since the gene knockout for B-myb has a lethal phenotype very early in development at the blastocyst stage, future studies in primary mouse megakaryocytes will require controlled ablation of the B-myb gene. We will describe preliminary studies on a new mouse line that we have created in which the B-myb gene has been modified to enable Cre-loxP mediated deletion.

PARP co-activates B-MYB through enhanced phosphorylation at cyclin/cdk2 sites.

PARP is a multifunctional protein that can affect genome stability, transcription control, telomere length and cell death. Recently we have reported that PARP binds to and enhances B-MYB transactivating potential. B-MYB is a potentially oncogenic transcription factor involved in mammalian cell proliferation, survival and differentiation. B-MYB gene expression is growth regulated and B-MYB protein is phosphorylated during S phase by cyclin A or E/cdk2 kinase, resulting in augmented transactivating potential. Here we show that PARP induces phosphorylation of B-MYB protein at cdk2 phosphorylation sites, since a B-MYB protein with mutated cdk2 phosphorylation sites is refractory to PARP-induced phosphorylation and co-activation in mammalian cells. We propose that PARP functions as a B-MYB co-factor by promoting cyclin/cdk2-dependent B-MYB phosphorylation. These results highlight a novel role for PARP as a factor that integrates cyclin-dependent kinases signaling with gene transcription.

B-Myb overexpression results in activation and increased Fas/Fas ligand-mediated cytotoxicity of T and NK cells.

The human B-myb gene encodes a transcriptional regulator that plays an important role in cell cycle progression, differentiation, and survival. To assess the in vivo role of B-myb, we investigated the phenotype of mouse transgenic lines in which B-Myb expression in lymphoid tissues was driven by the LCK proximal promoter. Overexpression of B-Myb had no measurable effect on the subsets of splenic and thymic lymphocytes, but was associated with increased expression of Fas ligand in NK and T cells. B-Myb-overexpressing splenocytes expressed higher IFN-gamma levels and contained higher percentages of cytokine-producing cells than wild-type (wt) splenocytes, as detected by Western blot analysis and ELISPOT assays, respectively. Ex vivo-cultured transgenic thymocytes and splenocytes had decreased survival compared with the corresponding cells from wt mice, possibly dependent on increased expression of Fas ligand. In addition, Fas ligand-dependent cytotoxicity of transgenic T and NK cells was significantly higher than that mediated by their wt counterparts. Together, these results indicate that B-Myb overexpression results in T and NK cell activation and increased cytotoxicity. Therefore, in addition to its well-established role in proliferation and differentiation, B-myb also appears to be involved in activation of NK and T cells and in their regulation of Fas/Fas ligand-mediated cytotoxicity

The Role of the MRG Gene Family in Replicative Senescence and Immortalization.

INTRODUCTION. Replicative senescence, the terminal loss of proliferation that normal cells undergo in vitro, is considered a model for aging at the cellular level. It is also considered a mechanism of tumor suppression because fusion of normal with immortal, tumor derived cell lines yields hybrids that regain growth control and cease division. Additional genetic analysis has revealed that a large number of immortal human cell lines assign to only four complementation groups for indefinite division (A-D) and microcell fusion studies have identified human chromosomes 1,4, and 7 as carrying the cell senescence gene loci for groups C, B and D respectively. We have cloned a gene on chromosome 4, MORF 4, that following transfection induces senescence only in immortal cell lines assigned to group B. MORF 4 is a member of a gene family MORF related genes (MRG) and the three expressed family members share interesting common motifs, including a leucine zipper region, a helix-loop-helix domain and nuclear localization signal. MRG 15 has a unique chromodomain at the amino terminus, MORF 4 has lost this region and MRG X has another unique amino terminal sequence with no known homology. We here present an update on our studies with these genes. METHODS. To identify proteins that interact with the MORF 4, MRG 15 and MRG X proteins we used the techniques of yeast two-hybrid, GST-pull down/Western, and immunoprecipitation (IP)/western analyses. Fractionation of nucleoprotein complexes was done on sucrose gradients. Transfection was performed using lipofectamine plus according to manufacturers instructions. RESULTS. We have identified a novel protein PAM (protein associated with MRG) that interacts with the MRG proteins. This identification was initially done by yeast two-hybrid analysis and confirmed by GST- pull-down and IP/Western experiments. We have also determined that MRG 15 and MRG X are in multiple nucleoprotein complexes. They cosediment on sucrose gradients with PAM and are present in the same immunoprecipitates. We are currently determining whether they are present in the same complex(es) or whether they interact with PAM in an independent complex(es), since yeast two-hybrid studies indicate they do not interact directly with each other. Both MRG X and MRG 15 can activate a promoterreporter construct of the b-myb gene and we are characterizing the region(s) of the proteins that is involved by the use of deletion mutants. We are also attempting to characterize the other components of the complex(es). DISCUSSION. Our studies with MRG X and MRG 15 indicate these proteins will be present in complexes that activate genes, and most likely be important in cell cycle progression. Since

An alternatively spliced isoform of B-Myb is a transcriptional inhibitor.

B-Myb is a highly conserved member of the Myb transcription factor family. The primary transcript of the B-myb gene is spliced alternatively in two mRNAs which either contain or lack a sequence corresponding to the so-called exon 9A of c-myb. Recent studies showed that full-length B-Myb containing the exon 9A encoded amino acids is a cell cycle regulated transcription factor whose activity is stimulated by cyclin A/Cdk 2-dependent phosphorylation at the carboxyl-terminus of B-Myb. We have now investigated in more detail the transactivation potential of the shorter isoform of B-Myb lacking exon 9A. Here, we show that B-Myb lacking exon 9A has no transactivation activity even in the presence of cyclin A. This inactivity of the shorter isoform of B-Myb is not due an improper subcelluar localization. Our work suggests that B-Myb lacking exon 9A may act as an inhibitor for full-length B-Myb mediated transactivation. Furthermore, by analysing the transactivation potential of Gal4/B-Myb fusion proteins we have identified the amino-terminal part of the exon 9A as the principal transactivation domain of full-length B-Myb. The results presented here demonstrate that B-myb encodes both an activator and an inhibitor of transcription and, thus, reveal an additional level of regulation of B-Myb activity beside the known cyclin dependent mechanisms.

Tax oncoprotein trans-represses endogenous B-myb promoter activity in human T cells.

The B-myb gene was identified on the basis of its homology with the protooncogene c-myb, homolog of the avian myeloblastosis virus (AMV) and avian leukemia virus (E26) transforming genes. Several studies using antisense constructs or antisense oligonucleotides as well as overexpression experiments suggest that B-Myb plays an important role in the transition from G(1) to S phase of the cell cycle and that B-Myb expression is cell cycle regulated. We have previously demonstrated that the human T cell lymphotropic virus type 1 (HTLV1) trans-activator Tax is able to repress transcription from c-myb promoter reporter constructs as well as from the endogenous c-myb promoter in human T cells and that this effect is mediated through inhibition of the c-Myb trans-activating functions. Here we report that both HTLV-1 as well as HTLV-2 Tax proteins inhibit c-Myb trans-activation in mouse embryo fibroblasts (MEFs). In addition to c-Myb, B-Myb expression is also markedly downregulated in HTLV-1-transformed cells at both RNA and protein levels. Furthermore, by using a Jurkat T cell line stably transfected with a tax gene driven by a cadmium-inducible promoter (JPX9), we were able to demonstrate that Tax directly represses the endogenous B-myb promoter in T cells. Because c-Myb and B-Myb have been involved in cell cycle progression, our results suggest that Tax, by repressing both c-Myb and B-Myb endogenous promoters, may bypass their requirement for cell cycle progression in HTLV-1-transformed T cells.

Physical interaction between CDK9 and B-Myb results in suppression of B-Myb gene autoregulation.

B-Myb is a transcription factor belonging to the myb family, whose activity has been associated with augmented DNA synthesis and cell cycle progression. We showed recently that B-Myb autoregulates its own expression through promoter transactivation. We report in this study that CDK9, the cyclin T associated kinase, which phosphorylates and activates RNA-Polymerase II, suppresses B-Myb autoregulation through direct interaction with the carboxyl-terminus of the B-Myb protein. Down-regulation of the transactivating ability of B-Myb is independent of the kinase activity of CDK9, because a kinase deficient mutant (dn-CDK9) also represses B-myb gene autoregulation. Overexpression of CDK9 did not result in suppression of p53-dependent transactivation or inhibition of the basal activity of the promoters tested so far, demonstrating that CDK9 is a B-Myb-specific repressor. Rather, transfection of the dominant negative dn-CDK9 construct inhibited the basal activity of the reporter genes, confirming an essential role for CDK9 in gene transcription. In addition, Cyclin T1 restores B-Myb transactivating activity when co-transfected along with CDK9, suggesting that the down-regulatory effect observed on B-Myb is specifically due to CDK9 alone. Thus, our data suggest that CDK9 is involved in the negative regulation of activated transcription mediated by certain transcription factors, such as B-Myb. This may indicate the existence of a feedback loop, mediated by the different activities of CDK9, which links basal with activated transcription.