The transcriptional coactivator MAML1 regulates p300 autoacetylation and HAT activity

MAML1 is a transcriptional coregulator originally identified as a Notch coactivator. MAML1 is also reported to interact with other coregulator proteins, such as CDK8 and p300, to modulate the activity of Notch. We, and others, previously showed that MAML1 recruits p300 to Notch-regulated genes through direct interactions with the DNA–CSL–Notch complex and p300. MAML1 interacts with the C/H3 domain of p300, and the p300–MAML1 complex specifically acetylates lysines of histone H3 and H4 tails in chromatin in vitro. In this report, we show that MAML1 potentiates p300 autoacetylation and p300 transcriptional activation. MAML1 directly enhances p300 HAT activity, and this coincides with the translocation of MAML1, p300 and acetylated histones to nuclear bodies

MAML1 acts cooperatively with EGR1 to activate EGR1-regulated promoters: implications for nephrogenesis and the development of renal cancer.

Mastermind-like 1 (MAML1) is a transcriptional coregulator of activators in various signaling pathways, such as Notch, p53, myocyte enhancer factor 2C (MEF2C) and beta-catenin. In earlier studies, we demonstrated that MAML1 enhanced p300 acetyltransferase activity, which increased the acetylation of Notch by p300. In this study, we show that MAML1 strongly induced acetylation of the transcription factor early growth response-1 (EGR1) by p300, and increased EGR1 protein expression in embryonic kidney cells. EGR1 mRNA transcripts were also upregulated in the presence of MAML1. We show that MAML1 physically interacted with, and acted cooperatively with EGR1 to increase transcriptional activity of the EGR1 and p300 promoters, which both contain EGR1 binding sites. Bioinformatics assessment revealed a correlation between p300, EGR1 and MAML1 copy number and mRNA alterations in renal clear cell carcinoma and p300, EGR1 and MAML1 gene alterations were associated with increased overall survival. Our findings suggest MAML1 may be a component of the transcriptional networks which regulate EGR1 target genes during nephrogenesis and could also have implications for the development of renal cell carcinoma

p300 and PCAF Act Cooperatively To Mediate Transcriptional Activation from Chromatin Templates by Notch Intracellular Domains In Vitro

Ligand activation of Notch receptors leads to release of the intracellular receptor domain (Notch IC), which translocates to the nucleus and interacts with the DNA-binding protein RBP-Jκ to control expression of specific target genes. A number of proteins have been shown to interact with Notch ICs and to modulate target gene activation, but the precise function of and interplay between these factors is not known. This report investigates the Notch IC-interacting proteins, p300, PCAF, and Mastermind-like 1 (MAML1), in an in vitro transcription system with purified factors and naked DNA or chromatin templates. MAML1, RBP-Jκ, and Notch IC are all required for optimal transcription from DNA, whereas transcription from chromatin requires, in addition, p300, which interacts with MAML1. The transcriptional activity of p300 requires acetyl coenzyme A, indicating that it functions as a histone acetyltransferase when mediating Notch IC function. PCAF is unable to promote transcription on its own but enhances Notch IC-mediated transcription from chromatin in conjunction with p300. These data define a critical role for p300 in the potentiation of Notch IC function by MAML1 and PCAF, provide the first evidence for cooperativity between PCAF and p300 in Notch IC function, and also indicate direct effects of RBP-Jκ, Notch IC, and MAML1 on the general transcription machinery

TRAP/SMCC/Mediator-Dependent Transcriptional Activation from DNA and Chromatin Templates by Orphan Nuclear Receptor Hepatocyte Nuclear Factor 4

The orphan nuclear receptor hepatocyte nuclear factor 4 (HNF-4) regulates the expression of many liver-specific genes both during development and in the adult animal. Towards understanding the molecular mechanisms by which HNF-4 functions, we have established in vitro transcription systems that faithfully recapitulate HNF-4 activity. Here we have focused on the coactivator requirements for HNF-4, especially for the multicomponent TRAP/SMCC/Mediator complex that has emerged as the central regulatory module of the transcription apparatus. Using a system that has been reconstituted from purified transcription factors, as well as one consisting of unfractionated nuclear extract from which TRAP/SMCC/Mediator has been depleted by specific antibodies, we demonstrate a strong dependence of HNF-4 function on this coactivator. Importantly, we further show a TRAP/SMCC/Mediator-dependence for HNF-4 transcriptional activation from chromatin templates. The latter involves cooperation with the histone acetyltransferase-containing coactivator p300, in accord with a synergistic mode of action of the two divergent coactivators. We also show that HNF-4 and TRAP/SMCC/Mediator can interact physically. This interaction likely involves primary HNF-4 activation function 2 (AF-2)-dependent interactions with the TRAP220 subunit of TRAP/SMCC/Mediator and secondary (AF-2-independent) interactions with TRAP170/RGR1. Finally, recruitment experiments using immobilized templates strongly suggest that the functional consequences of the physical interaction probably are manifested at a postrecruitment step in the activation pathway

Mastermind-Like 1 Is Ubiquitinated: Functional Consequences for Notch Signaling.

Early studies demonstrated the involvement of ubiquitination of the Notch intracellular domain for rapid turnover of the transcriptional complex at Notch target genes. It was shown that this ubiquitination was promoted by the co-activator Mastermind like 1 (MAML1). MAML1 also contains numerous lysine residues that may also be ubiquitinated and necessary for protein regulation. In this study, we show that over-expressed MAML1 is ubiquitinated and identify eight conserved lysine residues which are required for ubiquitination. We also show that p300 stimulates ubiquitination and that Notch inhibits ubiquitination. Furthermore, we show that a mutant MAML1 that has decreased ubiquitination shows increased output from a HES1 reporter gene assay. Therefore, we speculate that ubiquitination of MAML1 might be a mechanism to maintain low levels of the protein until needed for transcriptional activation. In summary, this study identifies that MAML1 is ubiquitinated in the absence of Notch signaling to maintain low levels of MAML1 in the cell. Our data supports the notion that a precise and tight regulation of the Notch pathway is required for this signaling pathway

Overview of Notch Signaling in the Nucleus.

<p>(A) In the absence of the NICD, Notch target genes remain in a repressed state through interaction of CBF1 with corepressor complexes (SMRT and HDAC1). (B) Release of the NICD from the cell membrane results in nuclear translocation and recruitment of MAML1, p300, and CDK8. CDK8 phosphorylates the NICD in the PEST domain as indicated by the lollipop structures. (C) Phosphorylation is thought to recruit the ubiquitin ligase Fbw7 to poly-ubiquitinate (boxes) the NICD, thereby signaling for degradation and shut off of target gene activation.</p

MAML1, CDK8 and p300 cooperatively stimulates Notch activity.

<p><b>(A)</b> HEK293 cells were co-transfected with a GAL4 luciferase gene and vectors expressing GAL4-Notch, MAML1, p300 and CDK8, as indicated in the figure. The data is presented as mean ± SD (n = 3). MAML1, p300, and CDK8 stimulate transcription in the presence of GAL4-Notch (*, p<0.0001). <b>(B)</b> Schematic of the <i>in vitro</i> transcription assay using chromatin templates. <b>(C)</b> Chromatin templates containing CSL binding sites were incubated with N1ICD, MAML1, CDK8, p300 and acetyl-CoA, as indicated.</p