Memory of past cellular responses is an essential adaptation to repeating environmental stimuli. We addressed the question of whether gamma interferon (IFN-␥)-inducible transcription generates memory that sensitizes cells to a second stimulus. We have found that the major histocompatibility complex class II gene DRA is relocated to promyelocytic leukemia (PML) nuclear bodies upon induction with IFN-␥, and this topology is maintained long after transcription shut off. Concurrent interaction of PML protein with mixedlineage leukemia generates a prolonged permissive chromatin state on the DRA gene characterized by high promoter histone H3 K4 dimethylation that facilitates rapid expression upon restimulation. We propose that the primary signal-induced transcription generates spatial and epigenetic memory that is maintained through several cell generations and endows the cell with increased responsiveness to future activation signals.
C.Spilianakis, A.Kretsovali and T.Agalioti contributed equally to this workWe describe the temporal order of recruitment of transcription factors, cofactors and basal transcriptional components and the consequent biochemical events that lead to activation of the major histocompatibility class II (MHCII) DRA gene transcription by IFN-g. We found that the gene is`poised' for activation since both the activators and a fraction of the basal transcriptional machinery are pre-assembled at the enhancer and promoter prior to IFN-g treatment. The class II transactivator is synthesized following IFN-g treatment and it is recruited to the enhanceosome leading to the subsequent recruitment of the CBP and GCN5 coactivators. This is followed by histone acetylation and recruitment of the SWI/SNF chromatin remodeling complex. CIITA also recruits the CDK7 and CDK9 kinases and enhances the ability of CDK7 to phosphorylate Pol II at Ser5 leading to initiation of mRNA synthesis. Thus, the gene-speci®c class II transactivator selects the target genes for expression by coordinating a multiple set of biochemical activities ranging from chromatin alterations and pre-initiation complex assembly to promoter clearance.
SummaryPromyelocytic leukemia protein (PML), the main constituent of PML nuclear bodies, regulates various physiological processes in different cell types. However, little is known about its functions in embryonic stem cells (ESC). Here, we report that PML contributes to ESC self-renewal maintenance by controlling cell-cycle progression and sustaining the expression of crucial pluripotency factors. Transcriptomic analysis and gain- or loss-of-function approaches showed that PML-deficient ESC exhibit morphological, metabolic, and growth properties distinct to naive and closer to the primed pluripotent state. During differentiation of embryoid bodies, PML influences cell-fate decisions between mesoderm and endoderm by controlling the expression of Tbx3. PML loss compromises the reprogramming ability of embryonic fibroblasts to induced pluripotent stem cells by inhibiting the transforming growth factor β pathway at the very early stages. Collectively, these results designate PML as a member of the regulatory network for ESC naive pluripotency and somatic cell reprogramming.
Class II transactivator (CIITA) is the master regulator of major histocompatibility complex class II genes that regulates both B lymphocyte-specific and interferon ␥-inducible expression. Here we identify protein regions and examine mechanisms that determine the intracellular distribution of CIITA. We show that two separate regions of CIITA mediate nuclear export: amino acids 1-114 and 408 -550. Both regions interact with the export receptor CRM-1. The CIITA region spanning amino acids 408 -550 of CIITA also determines its ability for homotypic self-association as well as heterotypic interactions with other regions residing at the amino and carboxyl termini of the protein. These observations are in line with data demonstrating that co-expression of amino-and carboxyl-terminal parts of CIITA promote subcellular relocalization and, remarkably, rescue transcriptional activation by individually inert molecules. CIITA point mutations that impair nuclear import and abolish its activation function show reduced self-association. We propose that the concerted action of homoand heterotypic interactions of CIITA determine proper protein configuration that in turn controls its nucleocytoplasmic trafficking. MHC1 class II genes are essential for the presentation of foreign antigens to T helper lymphocytes (1). These genes are specifically expressed in antigen-presenting cells and can be induced in various other cell types by cytokines such as interferon ␥ (2). In humans, the lack of MHC class II gene expression results in severe immunodeficiency, bare lymphocyte syndrome (3). Molecular analysis of the factors associated with this syndrome (RFX5, RFXAP, RFXANK, and CIITA) provided invaluable information about the regulation of MHC class II genes. Whereas the trimeric RFX and NFY complexes are ubiquitously expressed, expression of CIITA is cell type-restricted and correlates with constitutive or interferon ␥-mediated MHC class II gene expression (4 -6). CIITA is recruited on the class II promoter through multiple interactions with RFX, NFY, and possibly other factors (7-10). CIITA acts as a very potent transcriptional co-activator via its amino-terminal activation domain that can interact with components of the basal transcriptional machinery (11,12) and also with the histone acetylases CBP (13,14) and PCAF (15).CIITA has two nuclear localization signals (NLSs), one of which is localized at its carboxyl terminus and resides within a region deleted in a case of BLS (16), hereafter named NLS1. A second bipartite NLS (NLS2) resides at the amino-terminal part of the protein. Lysines within NLS2 are acetylated by PCAF and CBP (15). Nuclear levels of CIITA are regulated through many distinct mechanisms such as GTP binding to cognate sites (17), acetylation within the amino-terminal NLS2 (15), and protein-protein interactions involving the carboxylterminal leucine-rich repeats (8).Despite the two NLSs, CIITA is found both in the nucleus and cytoplasm (16, 17) because of active nuclear export (15). We show here that an amino-terminal ...
Transcriptional silencing during mitosis is caused by inactivation of critical transcriptional regulators and/or chromatin condensation. Inheritance of gene expression patterns through cell division involves various bookmarking mechanisms. In this report, we have examined the mitotic and post-mitotic expression of the DRA major histocompatibility class II (MHCII) gene in different cell types. During mitosis the constitutively MHCII-expressing B lymphoblastoid cells showed sustained occupancy of the proximal promoter by the cognate enhanceosome and general transcription factors. In contrast, although mitotic epithelial cells were depleted of these proteins irrespectively of their MHCII transcriptional activity, a distal enhancer selectively recruited the PP2A phosphatase via NFY and maintained chromatin accessibility. Based on our data, we propose a novel chromatin anti-condensation role for this element in mitotic bookmarking and timing of post-mitotic transcriptional reactivation.
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