David M. Heery

A signature motif in transcriptional co-activators mediates binding to nuclear receptors.

The binding of lipophilic hormones, retinoids and vitamins to members of the nuclear-receptor superfamily modifies the DNA-binding and transcriptional properties of these receptors, resulting in the activation or repression of target genes,. Ligand binding induces conformational changes in nuclear receptors and promotes their association with a diverse group of nuclear proteins, including SRC-1/p160 (refs 3-5), TIF-2/GRIP-1 (refs 6, 7) and CBP/p300 (refs 4, 5, 8, 9) which function as co-activators of transcription, and RIP-140 (ref. 10), TIF-1 (ref. 11) and TRIP-1/SUG-1 (refs 12, 13) whose functions are unclear. Here we report that a short sequence motif LXXLL (where L is leucine and X is any amino acid) present in RIP-140, SRC-1 and CBP is necessary and sufficient to mediate the binding of these proteins to liganded nuclear receptors. We show that the ability of SRC-1 to bind the oestrogen receptor and enhance its transcriptional activity is dependent upon the integrity of the LXXLL motifs and on key hydrophobic residues in a conserved helix (helix 12) of the oestrogen receptor that are required for its ligand-induced activation function. We propose that the LXXLL motif is a signature sequence that facilitates the interaction of different proteins with nuclear receptors, and is thus a defining feature of a new family of nuclear proteins.

The N-terminal part of TIF1, a putative mediator of the ligand-dependent activation function (AF-2) of nuclear receptors, is fused to B-raf in the oncogenic protein T18.

Nuclear receptors (NRs) bound to response elements mediate the effects of cognate ligands on gene expression. Their ligand‐dependent activation function, AF‐2, presumably acts on the basal transcription machinery through intermediary proteins/mediators. We have isolated a mouse nuclear protein, TIF1, which enhances RXR and RAR AF‐2 in yeast and interacts in a ligand‐dependent manner with several NRs in yeast and mammalian cells, as well as in vitro. Remarkably, these interactions require the amino acids constituting the AF‐2 activating domain conserved in all active NRs. Moreover, the oestrogen receptor (ER) AF‐2 antagonist hydroxytamoxifen cannot promote ER‐TIF1 interaction. We propose that TIF1, which contains several conserved domains found in transcriptional regulatory proteins, is a mediator of ligand‐dependent AF‐2. Interestingly, the TIF1 N‐terminal moiety is fused to B‐raf in the mouse oncoprotein T18.

Differential ligand-dependent interactions between the AF-2 activating domain of nuclear receptors and the putative transcriptional intermediary factors mSUG1 and TIF1.

Using a yeast two‐hybrid system we report the isolation of a novel mouse protein, mSUG1, that interacts with retinoic acid receptor alpha (RAR alpha) both in yeast cells and in vitro in a ligand‐ and AF‐2 activating domain (AF‐2 AD)‐dependent manner and show that it is a structural and functional homologue of the essential yeast protein SUG1. mSUG1 also efficiently interacts with other nuclear receptors, including oestrogen (ER), thyroid hormone (TR), Vitamin D3 (VDR) and retinoid X (RXR) receptors. By comparing the interaction properties of these receptors with mSUG1 and TIF1, we demonstrate that: (i) RXR alpha efficiently interacts with TIF1, but not with mSUG1, whereas TR alpha interacts much more efficiently with mSUG1 than with TIF1, and RAR alpha, VDR and ER efficiently interact with mSUG1 and TIF1; (ii) the amphipathic alpha‐helix core of the AF‐2 AD is differentially involved in interactions of RAR alpha with mSUG1 and TIF1; (iii) the AF‐2 AD cores of RAR alpha and ER are similarly involved in their interaction with TIF1, but not with mSUG1. Thus, the interaction interfaces between the different receptors and either mSUG1 or TIF1 may vary depending on the nature of the receptor and the putative mediator of its AF‐2 function. We discuss the possibility that mSUG1 and TIF1 may mediate the transcriptional activity of the AF‐2 of nuclear receptors through different mechanisms.

The AF1 and AF2 Domains of the Androgen Receptor Interact with Distinct Regions of SRC1

ABSTRACT The androgen receptor is unusual among nuclear receptors in that most, if not all, of its activity is mediated via the constitutive activation function in the N terminus. Here we demonstrate that p160 coactivators such as SRC1 (steroid receptor coactivator 1) interact directly with the N terminus in a ligand-independent manner via a conserved glutamine-rich region between residues 1053 and 1123. Although SRC1 is capable of interacting with the ligand-binding domain by means of LXXLL motifs, this interaction is not essential since an SRC1 mutant with no functional LXXLL motifs retains its ability to potentiate androgen receptor activity. In contrast, mutants lacking the glutamine-rich region are inactive, indicating that this region is both necessary and sufficient for recruitment of SRC1 to the androgen receptor. This recruitment is in direct contrast to the recruitment of SRC1 to the estrogen receptor, which requires interaction with the ligand-binding domain.

Global Histone Modifications in Breast Cancer Correlate with Tumor Phenotypes, Prognostic Factors, and Patient Outcome

Post-translational histone modifications are known to be altered in cancer cells, and loss of selected histone acetylation and methylation marks has recently been shown to predict patient outcome in human carcinoma. Immunohistochemistry was used to detect a series of histone lysine acetylation (H3K9ac, H3K18ac, H4K12ac, and H4K16ac), lysine methylation (H3K4me2 and H4K20me3), and arginine methylation (H4R3me2) marks in a well-characterized series of human breast carcinomas (n = 880). Tissue staining intensities were assessed using blinded semiquantitative scoring. Validation studies were done using immunofluorescence staining and Western blotting. Our analyses revealed low or absent H4K16ac in the majority of breast cancer cases (78.9%), suggesting that this alteration may represent an early sign of breast cancer. There was a highly significant correlation between histone modifications status, tumor biomarker phenotype, and clinical outcome, where high relative levels of global histone acetylation and methylation were associated with a favorable prognosis and detected almost exclusively in luminal-like breast tumors (93%). Moderate to low levels of lysine acetylation (H3K9ac, H3K18ac, and H4K12ac), lysine (H3K4me2 and H4K20me3), and arginine methylation (H4R3me2) were observed in carcinomas of poorer prognostic subtypes, including basal carcinomas and HER-2-positive tumors. Clustering analysis identified three groups of histone displaying distinct pattern in breast cancer, which have distinct relationships to known prognostic factors and clinical outcome. This study identifies the presence of variations in global levels of histone marks in different grades, morphologic types, and phenotype classes of invasive breast cancer and shows that these differences have clinical significance.

p38 MAPK/MK2-mediated induction of miR-34c following DNA damage prevents Myc-dependent DNA replication

The DNA damage response activates several pathways that stall the cell cycle and allow DNA repair. These consist of the well-characterized ATR (Ataxia telangiectasia and Rad-3 related)/CHK1 and ATM (Ataxia telangiectasia mutated)/CHK2 pathways in addition to a newly identified ATM/ATR/p38MAPK/MK2 checkpoint. Crucial to maintaining the integrity of the genome is the S-phase checkpoint that functions to prevent DNA replication until damaged DNA is repaired. Inappropriate expression of the proto-oncogene c-Myc is known to cause DNA damage. One mechanism by which c-Myc induces DNA damage is through binding directly to components of the prereplicative complex thereby promoting DNA synthesis, resulting in replication-associated DNA damage and checkpoint activation due to inappropriate origin firing. Here we show that following etoposide-induced DNA damage translation of c-Myc is repressed by miR-34c via a highly conserved target-site within the 3′ UTR. While miR-34c is induced by p53 following DNA damage, we show that in cells lacking p53 this is achieved by an alternative pathway which involves p38 MAPK signalling to MK2. The data presented here suggest that a major physiological target of miR-34c is c-Myc. Inhibition of miR-34c activity prevents S-phase arrest in response to DNA damage leading to increased DNA synthesis, DNA damage, and checkpoint activation in addition to that induced by etoposide alone, which are all reversed by subsequent c-Myc depletion. These data demonstrate that miR-34c is a critical regulator of the c-Myc expression following DNA damage acting downstream of p38 MAPK/MK2 and suggest that miR-34c serves to remove c-Myc to prevent inappropriate replication which may otherwise lead to genomic instability.

Analysis of the Steroid Receptor Coactivator 1 (SRC1)-CREB Binding Protein Interaction Interface and Its Importance for the Function of SRC1

ABSTRACT The transcriptional activity of nuclear receptors is mediated by coactivator proteins, including steroid receptor coactivator 1 (SRC1) and its homologues and the general coactivators CREB binding protein (CBP) and p300. SRC1 contains an activation domain (AD1) which functions via recruitment of CBP and and p300. In this study, we have used yeast two-hybrid and in vitro interaction-peptide inhibition experiments to map the AD1 domain of SRC1 to a 35-residue sequence potentially containing two α-helices. We also define a 72-amino-acid sequence in CBP necessary for SRC1 binding, designated the SRC1 interaction domain (SID). We show that in contrast to SRC1, direct binding of CBP to the estrogen receptor is weak, suggesting that SRC1 functions primarily as an adaptor to recruit CBP and p300. In support of this, we show that the ability of SRC1 to enhance ligand-dependent nuclear receptor activity in transiently transfected cells is dependent upon the integrity of the AD1 region. In contrast, the putative histone acetyltransferase domain, the Per-Arnt-Sim basic helix-loop-helix domain, the glutamine-rich domain, and AD2 can each be removed without loss of ligand-induced activity. Remarkably, a construct corresponding to residues 631 to 970, which contains only the LXXLL motifs and the AD1 region of SRC1, retained strong coactivator activity in our assays.

A highly conserved region in the hormone-binding domain of the human estrogen receptor functions as an efficient transactivation domain in yeast.

Human estrogen receptor (hER) mutants which activate transcription in the absence of hormone were isolated by random mutagenesis and genetic selection in the yeast Saccharomyces cerevisiae. Twenty constitutive hER mutants defining ten different alleles were selected. All sequence changes resulted in truncations of the receptor within a 123-amino-acid (aa) segment (aa 270 to 393) spanning the D region and the N-terminal part of region E which contains the hormone-binding domain (HBD). Transactivation assays using both the constitutive hER mutants and a series of deleted receptor derivatives generated in vitro revealed that the N-terminal part of region E, between aa 302 and 339, contains an efficient transcriptional activation function which is constitutively active in yeast. The location of this transactivation function in hER is similar to that of the tau 2 activation function of the glucocorticoid receptor and corresponds to a sequence which is highly conserved among the steroid hormone receptors. Thus, a conserved region exists in the HBD of the hER which can function as an autonomous transactivation domain.

Structural diversity in p160/CREB-binding protein coactivator complexes.

Ligand-induced transcription by nuclear receptors involves the recruitment of p160 coactivators such as steroid receptor coactivator 1 (SRC1), in complex with histone acetyltransferases such as CREB-binding protein (CBP) and p300. Here we describe the solution structure of a complex formed by the SRC1 interaction domain (SID) of CBP and the activation domain (AD1) of SRC1, both of which contain four helical regions (Cα1, Cα2, Cα3, and Cα3′ in CBP and Sα1, Sα2′, Sα2, and Sα3 in SRC1). A tight four-helix bundle is formed between Sα1, Cα1, Cα2, and Cα3 that is capped by Sα3. In contrast to the structure of the AD1 domain of the related p160 protein ACTR in complex with CBP SID, the sequences forming Sα2′ and Sα2 in SRC1 AD1 are not involved in the interface between the two domains but rather serve to position Sα3. Thus, although the CBP SID domain adopts a similar fold in complex with different p160 proteins, the topologies of the AD1 domains are strikingly different, a feature that is likely to contribute to functional specificity of these coactivator complexes.

An Extended LXXLL Motif Sequence Determines the Nuclear Receptor Binding Specificity of TRAP220

The interaction of coactivators with the ligand-binding domain of nuclear receptors (NRs) is mediated by amphipathic α-helices containing the signature motif LXXLL. TRAP220 contains two LXXLL motifs (LXM1 and LXM2) that are required for its interaction with NRs. Here we show that the nuclear receptor interaction domain (NID) of TRAP220 interacts weakly with Class I NRs. In contrast, SRC1 NID binds strongly to both Class I and Class II NRs. Interaction assays using nine amino acid LXXLL core motifs derived from SRC1 and TRAP220 revealed no discriminatory NR binding preferences. However, an extended LXM1 sequence containing amino acids −4 to +9, (where the first conserved leucine is +1) showed selective binding to thyroid hormone receptor and reduced binding to estrogen receptor. Replacement of either TRAP220 LXXLL motif with the corresponding 13 amino acids of SRC1 LXM2 strongly enhanced the interaction of the TRAP220 NID with the estrogen receptor. Mutational analysis revealed combinatorial effects of the LXM1 core and flanking sequences in the determination of the NR binding specificity of the TRAP220 NID. In contrast, a mutation that increased the spacing between TRAP220 LXM1 and LXM2 had little effect on the binding properties of this domain. Thus, a 13-amino acid sequence comprising an extended LXXLL motif acts as the key determinant of the NR binding specificity of TRAP220. Finally, we show that the NR binding specificity of full-length TRAP220 can be altered by swapping extended LXM sequences.