Tushar Chakraborty

Analysis of the myogenin promoter reveals an indirect pathway for positive autoregulation mediated by the muscle-specific enhancer factor MEF-2.

Transcriptional cascades that specify cell fate have been well described in invertebrates. In mammalian development, however, gene hierarchies involved in determination of cell lineage are not understood. With the recent cloning of the MyoD family of myogenic regulatory factors, a model system has become available with which to study the dynamics of muscle determination in mammalian development. Myogenin, along with other members of the MyoD gene family, possesses the apparent ability to redirect nonmuscle cells into the myogenic lineage. This ability appears to be due to the direct activation of an array of subordinate or downstream genes which are responsible for formation and function of the muscle contractile apparatus. Myogenin-directed transcription has been shown to occur through interaction with a DNA consensus sequence known as an E box (CANNTG) present in the control regions of numerous downstream genes. In addition to activating the transcription of subordinate genes, members of the MyoD family positively regulate their own expression and cross-activate one anothers expression. These autoregulatory interactions have been suggested as a mechanism for induction and maintenance of the myogenic phenotype, but the molecular details of the autoregulatory circuits are undefined. Here we show that the myogenin promoter contains a binding site for the myocyte-specific enhancer-binding factor, MEF-2, which can function as an intermediary of myogenin autoactivation. Since MEF-2 can be induced by myogenin, these results suggest that myogenin and MEF-2 participate in a transcriptional cascade in which MEF-2, once induced by myogenin, acts to amplify and maintain the myogenic phenotype by acting as a positive regulator of myogenin expression.

The basic region of myogenin cooperates with two transcription activation domains to induce muscle-specific transcription.

Myogenin is a skeletal muscle-specific transcription factor that can activate myogenesis when introduced into a variety of nonmuscle cell types. Activation of the myogenic program by myogenin is dependent on its binding to a DNA sequence known as an E box, which is associated with numerous muscle-specific genes. Myogenin shares homology with MyoD and other myogenic regulatory factors within a basic region and a helix-loop-helix (HLH) motif that mediate DNA binding and dimerization, respectively. Here we show that the basic region-HLH motif of myogenin alone lacks transcriptional activity and is dependent on domains in the amino and carboxyl termini to activate transcription. Analysis of these N- and C-terminal domains through creation of chimeras with the DNA-binding domain of the Saccharomyces cerevisiae transcription factor GAL4 revealed that they act as strong transcriptional activators. These transcription activation domains are dependent for activity on a specific amino acid sequence within the basic region, referred to as the myogenic recognition motif (MRM), when an E box is the target for DNA binding. However, the activation domains function independent of the MRM when DNA binding is mediated through a heterologous DNA-binding domain. The activation domain of the acidic coactivator VP16 can substitute for the myogenin activation domains and restore strong myogenic activity to the basic region-HLH motif. Within a myogenin-VP16 chimera, however, the VP16 activation domain also relies on the MRM for activation of the myogenic program. These findings reveal that DNA binding and transcriptional activation are separable functions, encoded by different domains of myogenin, but that the activity of the transcriptional activation domains is influenced by the DNA-binding domain. Activation of muscle-specific transcription requires collaboration between the DNA-binding and activation domains of myogenin and is dependent on events in addition to DNA binding.

Molecular control of myogenesis: antagonism between growth and differentiation

Insight into the molecular mechanisms that control establishment of the skeletal muscle phenotype has recently been obtained through cloning of a family of muscle-specific regulatory factors that can activate myogenesis when transfected into non-muscle cells. This family of factors, which includes MyoD, myogenin, myf-5, and MRF4, can bind DNA and transactivate muscle-specific genes in collaboration with ubiquitous cellular factors. Growth factors play an antagonistic role in myogenesis by suppressing the actions of the myogenic regulatory factor family. This review will focus on the regulation and mechanism of action of this family of myogenic regulatory factors and on the central role of peptide growth factors in modulating their expression and biological activities.

Inefficient homooligomerization contributes to the dependence of myogenin on E2A products for efficient DNA binding.

Myogenin is a muscle-specific transcription factor that can activate myogenesis; it belongs to a family of transcription factors that share homology within a basic region and an adjacent helix-loop-helix (HLH) motif. Although myogenin alone binds DNA inefficiently, in the presence of the widely expressed HLH proteins E12 and E47 (encoded by the E2A gene), it forms heterooligomers that bind with high affinity to a DNA sequence known as a kappa E-2 site. In contrast, E47 and to a lesser extent E12 are both able to bind the kappa E-2 site relatively efficiently as homooligomers. To define the relative contributions of the basic regions of myogenin and E12 to DNA binding and muscle-specific gene activation, we created chimeras of the two proteins by swapping their basic regions. We showed that myogenins weak affinity for the kappa E-2 site is attributable to inefficient homooligomerization and that the myogenin basic domain alone can mediate high-affinity DNA binding when placed in E12. Within a heterooligomeric complex, two basic regions were required to form a high-affinity DNA-binding domain. Basic-domain mutants of myogenin or E2A gene products that cannot bind DNA retained the ability to oligomerize and could abolish DNA binding of the wild-type proteins in vitro. These myogenin and E2A mutants also acted as trans-dominant inhibitors of muscle-specific gene activation in vivo. These findings support the notion that muscle-specific gene activation requires oligomerization between myogenin and E2A gene products and that E2A gene products play an important role in myogenesis by enhancing the DNA-binding activity of myogenin, as well as other myogenic HLH proteins.

Domains outside of the DNA-binding domain impart target gene specificity to myogenin and MRF4.

Myogenin and MRF4 belong to the MyoD family of muscle-specific transcription factors, which can activate myogenesis when introduced into nonmyogenic cells. These proteins share homology within a basic-helix-loop-helix motif that mediates DNA binding and dimerization, but they are divergent in their amino and carboxyl termini. Although myogenin and MRF4 bind the same sequence within the muscle creatine kinase enhancer, only myogenin efficiently transactivates this enhancer. By creating chimeras of myogenin and MRF4, we show that the specificities of these factors for transactivation of the muscle creatine kinase enhancer can be interchanged by swapping their amino and carboxyl termini. Within these chimeras, strong cooperation between the amino and carboxyl termini was observed. These findings suggest that myogenin and MRF4 discriminate between muscle-specific enhancers and that target gene specificity is determined by domains surrounding the basic-helix-loop-helix region.

High Reactivation of BK Virus Variants in Asian Indians with Renal Disorders and During Pregnancy

There is resurgence of interest in the study of occurrence, genotype and pathogenic associations of human Polyomavirus BK and JC in recent years. In the present study, we have ascertained the presence of BK virus shed in the urine samples of pregnant women and immunocompromised patients, for the first time in Asian Indian population, and have also characterised the prevalent genotypes of the non-coding control regions (NCCRs) of these natural isolates. The results strongly suggest a very high incidence, as well as degree, of BK virus reactivation in this population groups assayed. Approximately 65% of the patients and pregnant women together, tested positive based on polymerase chain reaction (PCR) analysis, and these results were further confirmed by Southern hybridisation and dot blot against BKV specific probes. The NCCRs of the several Indian endemic strains were analysed by sequencing PCR products, amplified directly from urine samples, with oligonucleotide primers designed from the constant region of T-Antigen and VP2 coding sequences. The typical features of the NCCRs of these Indian strains appeared to be comparable and related to the archetypal strain BKV (WW) with some alterations in few key positions. Apart from these subtle alterations, neither any major DNA rearrangement within the NCCR region nor any drastic modification marked BKV strains found in nephropathy and in the healthy subjects (pregnancy). However, in some of the immunocompromised patients studied, the degree of reactivations reflected by viruria, appeared to be much higher compared to other reports.

Identification of HeLa cell nuclear factors that bind to and activate the early promoter of human polyomavirus BK in vitro.

Human polyomavirus BK (BKV), an oncogenic DNA virus, differs from other papovaviruses in the organization of the regulatory region and in tissue tropism for kidney cells. The noncoding regulatory region of the viral genome in prototype strains includes three 68-base-pair (bp) repeats, each containing a number of potential regulatory elements. Some of these signals are unique to human papovaviruses, and others are homologous to those identified in many viral and cellular genes. We evaluated the contribution of individual 68-bp repeats to the initiation of transcription from the early promoter in a HeLa cell extract and identified cis-acting elements to which human cellular factors bind to activate transcription. The early promoter with only one copy of the 68-bp repeat could accurately initiate transcription in vitro, but additional copies were required for its stimulation. DNA-binding assays and DNase I protection experiments identified six domains in the regulatory region protected by human cellular factors. Two of these footprints were located within the proximal and distal 68-bp repeats, and one was located at the late side of the repeats. These footprints were centered over a TGGA(N)5-6GCCA core and were produced by a protein of the nuclear factor 1 (NF-1) family. This protein is either identical or similar to that which binds to the high-affinity site at the origin of adenovirus DNA replication. Three other domains, two at the junctions of the 68-bp repeats and one in the late side of the repeats, were partially protected by proteins with AP-1- and Sp-1-like activities. Transcription initiation from the early promoter was drastically reduced when a complete 68-bp repeat or the NF-1 binding site was used as a competitor in the in vitro assay. However, a point mutation within the NF-1 binding site, which reduced NF-1 binding in vitro to a level comparable to that of nonspecific DNA, also eliminated its ability to compete with early transcription. The murine homolog of the AP-1 binding site had a modest effect on in vitro transcription. Our results suggest that, among the multiple HeLa cell nuclear factors, NF-1 acts as a major activator of the early promoter in vitro.