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Differential regulation of mouse germline Ig gamma 1 and epsilon promoters by IL-4 and CD40Before Ig class switching, RNA transcription through the specific S regions undergoing recombination is induced by cytokines and other activators that induce and direct switching. The resulting germline (GL) transcripts are essential for switch recombination. To understand the differential regulation of mouse IgG1 and IgE, we compared the promoters for GL gamma1 and epsilon transcripts. We addressed the question of why the promoter that regulates GL epsilon transcription is more responsive to IL-4 than the gamma1 promoter and also why GL epsilon transcription is more dependent on IL-4 than is gamma1 transcription. We found that the IL-4-responsive region of the GL epsilon promoter is more inducible than that of the gamma1 promoter, although each promoter contains a binding site for the IL-4-inducible transcription factor Stat6, located immediately adjacent to a binding site for a basic region leucine zipper (bZip) family protein. However, the arrangement and sequences of the sites differ between the epsilon and gamma1 promoters. The GL epsilon promoter binds Stat6 with a 10-fold higher affinity than does the gamma1 promoter. Furthermore, the bZip elements of the two promoters bind different transcription factors, as the GL epsilon promoter binds and is activated by AP-1, whereas the gamma1 promoter binds and is activated by activating transcription factor 2. C/EBPbeta and C/EBPgamma also bind the gamma1 bZip element, although they inhibit rather than activate transcription. However, inhibition of promoter activity by C/EBPbeta does not require the bZip element and may instead occur via inhibiting the activity of NF-kappaB.
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Genome-wide functional analysis reveals factors needed at the transition steps of induced reprogrammingAlthough transcriptome analysis can uncover the molecular changes that occur during induced reprogramming, the functional requirements for a given factor during stepwise cell-fate transitions are left unclear. Here, we used a genome-wide RNAi screen and performed integrated transcriptome analysis to identify key genes and cellular events required at the transition steps in reprogramming. Genes associated with cell signaling pathways (e.g., Itpr1, Itpr2, and Pdia3) constitute the major regulatory networks before cells acquire pluripotency. Activation of a specific gene set (e.g., Utf1 or Tdgf1) is important for mature induced pluripotent stem cell formation. Strikingly, a major proportion of RNAi targets ( approximately 53% to 70%) includes genes whose expression levels are unchanged during reprogramming. Among these non-differentially expressed genes, Dmbx1, Hnf4g, Nobox, and Asb4 are important, whereas Nfe2, Cdkn2aip, Msx3, Dbx1, Lzts1, Gtf2i, and Ankrd22 are roadblocks to reprogramming. Together, our results provide a wealth of information about gene functions required at transition steps during reprogramming.
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The human homologue of the yeast DNA repair and TFIIH regulator MMS19 is an AF-1-specific coactivator of estrogen receptorSteroid/nuclear hormone receptors are ligand-dependent transcriptional regulators that control gene expression in a wide array of biological processes. The transcriptional activity of the receptors is mediated by an N-terminal ligand-independent transcriptional activation function AF-1 and a C-terminal ligand-dependent transcriptional activation function AF-2. The nuclear receptor coactivator RAC3 (also known as AIB1/ACTR/pCIP/TRAM-1/SRC-3) is amplified in breast cancer cells, where it forms a complex with estrogen receptor (ER) and enhances AF-2 activity of the receptor. Here, we identify a putative human homologue of the yeast DNA repair and transcriptional regulator MMS19 as a RAC3-interacting protein. The human MMS19 interacts with the N-terminal PAS-A/B domain of RAC3 in vivo and in vitro through a conserved C-terminal domain. Interestingly, the human MMS19 also interacts with estrogen receptors in a ligand-independent manner but not with retinoic acid receptor or thyroid hormone receptor. Overexpression of the interacting domain of hMMS19 strongly inhibits ER-mediated transcriptional activation, indicating a dominant negative activity. In contrast, over expression of the full-length hMMS19 enhances ER-mediated transcriptional activation. We find that hMMS19 stimulates the AF-1 activity of ERalpha, but not the AF-2 activity, suggesting that hMMS19 may be an AF-1-specific transcriptional coactivator of estrogen receptor.
