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Subcellular partitioning of transcription factors during osteoblast differentiation: developmental association of the AML/CBF alpha/PEBP2 alpha-related transcription factor-NMP-2 with the nuclear matrixThe subnuclear location of transcription factors may functionally contribute to the regulation of gene expression. Several classes of gene regulators associate with the nuclear matrix in a cell type, cell growth, or cell cycle related-manner. To understand control of nuclear matrix-transcription factor interactions during tissue development, we systematically analyzed the subnuclear partitioning of a panel of transcription factors (including NMP-1/YY-1, NMP-2/AML, AP-1, and SP-1) during osteoblast differentiation using biochemical fractionation and gel shift analyses. We show that nuclear matrix association of the tissue-specific AML transcription factor NMP-2, but not the ubiquitous transcription factor YY1, is developmentally upregulated during osteoblast differentiation. Moreover, we show that there are multiple AML isoforms in mature osteoblasts, consistent with the multiplicity of AML factors that are derived from different genes and alternatively spliced cDNAs. These AML isoforms include proteins derived from the AML-3 gene and partition between distinct subcellular compartments. We conclude that the selective partitioning of the YY1 and AML transcription factors with the nuclear matrix involves a discriminatory mechanism that targets different classes and specific isoforms of gene regulatory factors to the nuclear matrix at distinct developmental stages. Our results are consistent with a role for the nuclear matrix in regulating the expression of bone-tissue specific genes during development of the mature osteocytic phenotype.
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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.
