Maf1 is a negative regulator of transcription in Trypanosoma brucei.

RNA polymerase III (Pol III) produces small RNA molecules that play essential roles in mRNA processing and translation. Maf1, originally described as a negative regulator of Pol III transcription, has been studied from yeast to human. Here we characterized Maf1 in the parasitic protozoa Trypanosoma brucei (TbMaf1), representing the first report to analyse Maf1 in an early-diverged eukaryote. While Maf1 is generally encoded by a single-copy gene, the T. brucei genome contains two almost identical TbMaf1 genes. The TbMaf1 protein has the three conserved sequences and is predicted to fold into a globular structure. Unlike in yeast, TbMaf1 localizes to the nucleus in procyclic forms of T. brucei under normal growth conditions. Cell lines that either downregulate or overexpress TbMaf1 were generated, and growth curve analysis with them suggested that TbMaf1 participates in the regulation of cell growth of T. brucei. Nuclear run-on and chromatin immunoprecipitation analyses demonstrated that TbMaf1 represses Pol III transcription of tRNA and U2 snRNA genes by associating with their promoters. Interestingly, 5S rRNA levels do not change after TbMaf1 ablation or overexpression. Notably, our data also revealed that TbMaf1 regulates Pol I transcription of procyclin gene and Pol II transcription of SL RNA genes.

MafA is a dedicated activator of the insulin gene in vivo.

As successful generation of insulin-producing cells could be used for diabetes treatment, a concerted effort is being made to understand the molecular programs underlying islet beta-cell formation and function. The closely related MafA and MafB transcription factors are both key mammalian beta-cell regulators. MafA and MafB are co-expressed in insulin+beta-cells during embryogenesis, while in the adult pancreas only MafA is produced in beta-cells and MafB in glucagon+alpha-cells. MafB-/- animals are also deficient in insulin+ and glucagon+ cell production during embryogenesis. However, only MafA over-expression selectively induced endogenous Insulin mRNA production in cell line-based assays, while MafB specifically promoted Glucagon expression. Here, we analyzed whether these factors were sufficient to induce insulin+ and/or glucagon+ cell formation within embryonic endoderm using the chick in ovo electroporation assay. Ectopic expression of MafA, but not MafB, promoted Insulin production; however, neither MafA nor MafB were capable of inducing Glucagon. Co-electroporation of MafA with the Ngn3 transcription factor resulted in the development of more organized cell clusters containing both insulin- and glucagon-producing cells. Analysis of chimeric proteins of MafA and MafB demonstrated that chick Insulin activation depended on sequences within the MafA C-terminal DNA-binding domain. MafA was also bound to Insulin and Glucagon transcriptional control sequences in mouse embryonic pancreas and beta-cell lines. Collectively, these results demonstrate a unique ability for MafA to independently activate Insulin transcription.

Multiple mechanisms and functions of maf transcription factors in the regulation of tissue-specific genes.

Maf family transcription factors are regulators of tissue-specific gene expression and cell-differentiation in a wide variety of tissues and are also involved in human diseases and oncogenic transformation. To establish tissue-specific expression, Maf binds to Maf-recognition elements (MAREs) in the regulatory regions of target genes, and functionally interacts with other transcription factors. For example, L-Maf and c-Maf, which are specifically expressed in developing lens cells, act synergistically with Sox proteins to induce lens-specific crystalline genes. MafA, a beta-cell-specific member of the Maf family, activates the insulin gene promoter synergistically with Pdx1 and Beta2 to establish beta-cell specific expression. Furthermore, in beta-cells, MafA activity is regulated at both the transcriptional and post-translational levels by glucose and oxidative stress. This review summarizes the functions and roles of Maf in various biological processes and recent progress in elucidating the mechanisms whereby Maf proteins regulate transcription.

L-Maf regulates p27kip1 expression during chick lens fiber differentiation.

Organ formation requires spatio-temporal proliferation and differentiation of precursor cells. During lens development, placodal cells in the posterior lens vesicle exit from the cell cycle and enter into the process of differentiation. Cyclin-dependent kinase inhibitors play critical roles in cell cycle exit and promote differentiation in several tissues. We have found that p27kip1 is expressed in the posterior lens cells that undergo differentiation to form the differentiated fiber cells. The transcription factor L-Maf is expressed in these cells earlier than p27kip1. From in ovo gain- or loss-of-function experiments, we have found that L-Maf can, respectively, induce or inhibit the expression of p27kip1 in lens cells. Promoter assays using the 5' upstream sequences of the human p27kip1 gene indicate that L-Maf can activate p27kip1 transcription through the basal regulatory region. We suggest that L-Maf regulates cell cycle exit of the posterior lens cells by activating p27kip1, and thus directs fiber cell differentiation during lens formation in chick.

Sequential and combinatorial roles of maf family genes define proper lens development.

PURPOSE: Maf proteins have been shown to play pivotal roles in lens development in vertebrates. The developing chick lens expresses at least three large Maf proteins. However, the transcriptional relationship among the three large maf genes and their various roles in transactivating the downstream genes largely remain to be elucidated. METHODS: Chick embryos were electroporated with wild-type L-maf, c-maf, and mafB by in ovo electroporation, and their effects on gene expression were determined by in situ hybridization using specific probes or by immunostaining. Endogenous gene expression was determined using nonelectroporated samples. RESULTS: A regulation mechanism exists among the members of maf family gene. An early-expressed member of this gene family typically stimulates the expression of later-expressed members. We also examined the regulation of various lens-expressing genes with a focus on the interaction between different Maf proteins. We found that the transcriptional ability of Maf proteins varies, even when the target is the same, in parallel with their discrete functions. L-Maf and c-Maf have no effect on E-cadherin expression, whereas MafB enhances its expression and thereby impedes lens vesicle formation. This study also revealed that Maf proteins can regulate the expression of gap junction genes, connexins, and their interacting partner, major intrinsic protein (MIP), during lens development. Misexpression of L-Maf and c-Maf induces ectopic expression of Cx43 and MIP; in contrast, MafB appears to have no effect on Cx43, but induces MIP significantly as evidenced from our gain-of-function experiments. CONCLUSIONS: Our results indicate that large Maf function is indispensable for chick lens initiation and development. In addition, L-Maf positively regulates most of the essential genes in this program and directs a series of molecular events leading to proper formation of the lens.

Transcription factors regulating beta-cell function.

Type 2 diabetes is primarily associated with insulin resistance and beta-cell dysfunction. Maintenance of functional mature beta-cells is imperative for ensuring glucose homeostasis. This can be achieved by optimal expression of key transcription factors that are required for normal pancreatic development and maintaining beta-cell function. Defining the regulation of transcription factors as well as their regulation of important beta-cell genes like insulin will provide further insight into elucidating the mechanisms leading to beta-cell dysfunction.