The Mediator subunit MED1 is essential for mammary gland development and lactation, whose contribution through direct interaction with estrogen receptors (ERs) is restricted to involvement in pubertal mammary gland development and luminal cell differentiation. Here, we provide evidence that the MED24-containing submodule of Mediator functionally communicates specifically with MED1 in pubertal mammary gland development. Mammary glands from MED1/MED24 double heterozygous knockout mice showed profound retardation in ductal branching during puberty, while single haploinsufficient glands developed normally. DNA synthesis of both luminal and basal cells were impaired in double mutant mice, and the expression of ERtargeted genes encoding E2F1 and cyclin D1, which promote progression through the G 1 /S phase of the cell cycle, was attenuated. Luciferase reporter assays employing double mutant mouse embryonic fibroblasts showed selective impairment in ER functions. Various breast carcinoma cell lines expressed abundant amounts of MED1, MED24, and MED30, and attenuated expression of MED1 and MED24 in breast carcinoma cells led to attenuated DNA synthesis and growth. These results indicate functional communications between the MED1 subunit and the MED24-containing submodule that mediate estrogen receptor functions and growth of both normal mammary epithelial cells and breast carcinoma cells. N uclear receptors, which include steroid and nonsteroid hormone receptors, comprise a superfamily of DNA-bound transcriptional regulators that are activated in response to specific small lipophilic ligands and that play major physiological roles in cell growth, differentiation, and homeostasis (reviewed in references 10 and 25). Estrogen receptor ␣ (ER␣) is the key activator that leads to growth of the mammary glands during adolescence, as well as during pregnancy, in response to elevated plasma estrogen levels. Among the hormone-responsive genes transcribed under the control of ER␣ is another steroid hormone receptor, progesterone receptor (PR), which in concert with ER␣, plays an important role in mammary gland development (5).The metazoan Mediator/TRAP coactivator complex is a master transcriptional coregulator composed of about 30 subunits and is structurally subdivided into head, body, and tail modules. It constitutes a subcomplex of the RNA polymerase II holoenzyme and integrates a wide variety of intracellular signals through specific interactions of activators with specific Mediator subunits that reside predominantly at its tail module (reviewed in references 4, 6, 15, 20, and 24). We have proposed a multistep model for nuclear receptor-induced transcriptional activation (15). In this model, histone-modifying coactivators that possess either histone acetyltransferase or histone methyltransferase activities first interact with ligand-bound nuclear receptors, and chromatin structure is subsequently relaxed. Then an exchange of coactivators takes place and the Mediator is bound to nuclear receptors through two canonical LxxLL nuc...

The rare actinomycete Actinoplanes missouriensis forms sporangia, including hundreds of flagellated spores that start swimming as zoospores after their release. Under conditions suitable for vegetative growth, zoospores stop swimming and germinate. A comparative proteome analysis between zoospores and germinating cells identified 15 proteins that were produced in larger amounts in germinating cells. They include an orthologue of BldD (herein named AmBldD [BldD of A. missouriensis]), which is a transcriptional regulator involved in morphological development and secondary metabolism in Streptomyces. AmBldD was detected in mycelia during vegetative growth but was barely detected in mycelia during the sporangiumforming phase, in spite of the constant transcription of AmbldD throughout growth. An AmbldD mutant started to form sporangia much earlier than the wild-type strain, and the resulting sporangia were morphologically abnormal. Recombinant AmBldD bound a palindromic sequence, the AmBldD box, located upstream from AmbldD. 3=,5=-Cyclic di-GMP significantly enhanced the in vitro DNA-binding ability of AmBldD. A chromatin immunoprecipitation-sequencing analysis and an in silico search for AmBldD boxes revealed that AmBldD bound 346 genomic loci that contained the 19-bp inverted repeat 5=-NN(G/A)TNACN(C/G)N(G/C)NGTNA(C/T)NN-3= as the consensus AmBldDbinding sequence. The transcriptional analysis of 27 selected AmBldD target gene candidates indicated that AmBldD should repress 12 of the 27 genes, including bldM, ssgB, whiD, ddbA, and wblA orthologues. These genes are involved in morphological development in Streptomyces coelicolor A3(2). Thus, AmBldD is a global transcriptional regulator that seems to repress the transcription of tens of genes during vegetative growth, some of which are likely to be required for sporangium formation.IMPORTANCE The rare actinomycete Actinoplanes missouriensis undergoes complex morphological differentiation, including sporangium formation. However, almost no molecular biological studies have been conducted on this bacterium. BldD is a key global regulator involved in the morphological development of streptomycetes. BldD orthologues are highly conserved among sporulating actinomycetes, but no BldD orthologues, except one in Saccharopolyspora erythraea, have been studied outside the streptomycetes. Here, it was revealed that the BldD orthologue AmBldD is essential for normal developmental processes in A. missouriensis. The AmBldD regulon seems to be different from the BldD regulon in Streptomyces coelicolor A3(2), but they share four genes that are involved in morphological differentiation in S. coelicolor A3(2).KEYWORDS gene regulation, rare actinomycete, regulon, sporangium formation, transcriptional factor A ctinomycetes are Gram-positive, mainly soil-inhabiting bacteria. Many genera of actinomycetes show filamentous growth and produce spores, and therefore, actinomycetes are generally characterized by a complex morphological development. For

The MED1 subunit of the Mediator transcriptional coregulator complex coactivates GATA1 and induces erythropoiesis. Here, we show the dual mechanism of GATA1- and MED1-mediated transcription. MED1 expression levels in K562 erythroleukemia cells paralleled the levels of GATA1-targeted gene transcription and erythroid differentiation. An N-terminal fragment of MED1, MED1(1–602), which is incapable of interacting with GATA1, enhanced GATA1-targeted gene transcription and erythroid differentiation, and introduction of MED1(1–602) into Med1−/− mouse embryonic fibroblasts (MEFs) partially rescued GATA1-mediated transcription. The C-terminal zinc-finger domain of GATA1 interacts with the MED1(1–602)-interacting coactivator CCAR1, CoCoA, and MED1(681–715). CCAR1 and CoCoA synergistically enhanced GATA1-mediated transcription from the γ-globin promoter in MEFs. Recombinant GATA1, CCAR1, CoCoA, and MED1(1–602) formed a complex in vitro, and GATA1, CCAR1, CoCoA, and MED1 were recruited to the γ-globin promoter in K562 cells during erythroid differentiation. Therefore, in addition to the direct interaction between GATA1 and MED1, CoCoA and CCAR1 appear to relay the GATA1 signal to MED1, and multiple modes of the GATA1-MED1 axis may help to fine-tune GATA1 function during GATA1-mediated homeostasis events.

The rare actinomycete Actinoplanes missouriensis forms terminal sporangia containing a few hundred flagellated spores. In response to water, the sporangia open and release the spores into external environments. The orphan response regulator TcrA functions as a global transcriptional activator during sporangium formation and dehiscence. Here, we report the characterization of an orphan hybrid histidine kinase, HhkA. Sporangia of the hhkA-deleted mutant contained many distorted or ectopically germinated spores and scarcely opened to release the spores under sporangium dehiscence-inducing conditions. These phenotypic changes are quite similar to those observed in the tcrA-deleted mutant. Comparative RNA sequencing analysis showed that genes controlled by HhkA mostly overlap with TcrA-regulated genes. The direct interaction between HhkA and TcrA was suggested by a bacterial two-hybrid assay, but not conclusive. The phosphorylation of TcrA using acetyl phosphate as a phosphate donor markedly enhanced its affinity to the TcrA box sequences in the electrophoretic mobility shift assay. Together with other results, we proposed that HhkA and TcrA comprise a cognate two-component regulatory system, which controls the transcription of the genes involved in many aspects of morphological development including sporangium formation, spore dormancy, and sporangium dehiscence in A. missouriensis. IMPORTANCE Actinoplanes missouriensis goes through complex morphological differentiation, including flagellated spore-containing sporangium formation, sporangium dehiscence, swimming of zoospores, and germination of zoospores to filamentous growth. Although the orphan response regulator TcrA globally activates many genes required for sporangium formation, spore dormancy, and sporangium dehiscence, its partner histidine kinase remained unclear. Here, we analyzed the function of an orphan hybrid histidine kinase, HhkA, and proposed that HhkA constitutes a cognate two-component regulatory system with TcrA. That HhkA and TcrA homologues are highly conserved among the genus Actinoplanes and several closely related rare actinomycetes indicates that this possible two-component regulatory system has been employed for complex morphological development in sporangium- and/or zoospore-forming rare actinomycetes.

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