Robert G. Roeder

Activation of p53 Sequence-Specific DNA Binding by Acetylation of the p53 C-Terminal Domain

The tumor suppressor p53 exerts antiproliferation effects through its ability to function as a sequence-specific DNA-binding transcription factor. Here, we demonstrate that p53 can be modified by acetylation both in vivo and in vitro. Remarkably, the site of p53 that is acetylated by its coactivator, p300, resides in a C-terminal domain known to be critical for the regulation of p53 DNA binding. Furthermore, the acetylation of p53 can dramatically stimulate its sequence-specific DNA-binding activity, possibly as a result of an acetylation-induced conformational change. These observations clearly indicate a novel pathway for p53 activation and, importantly, provide an example of an acetylation-mediated change in the function of a nonhistone regulatory protein. These results have significant implications regarding the molecular mechanisms of various acetyltransferase-containing transcriptional coactivators whose primary targets have been presumed to be histones.

The role of general initiation factors in transcription by RNA polymerase II

Transcription initiation on protein-encoding genes represents a major control point for gene expression in eukaryotes, and is mediated by RNA polymerase II and a surprisingly complex array of general initiation factors (TFIIA, -B, -D, -E, -F and -H) that are highly conserved from yeast to man. Elucidation of structural and functional features of these factors on model promoters has revealed insights into biochemical mechanisms and provides a basis for understanding their regulation on diverse promoters by gene- and cell-specific activators.

Transient high glucose causes persistent epigenetic changes and altered gene expression during subsequent normoglycemia

The current goal of diabetes therapy is to reduce time-averaged mean levels of glycemia, measured as HbA1c, to prevent diabetic complications. However, HbA1c only explains <25% of the variation in risk of developing complications. Because HbA1c does not correlate with glycemic variability when adjusted for mean blood glucose, we hypothesized that transient spikes of hyperglycemia may be an HbA1c–independent risk factor for diabetic complications. We show that transient hyperglycemia induces long-lasting activating epigenetic changes in the promoter of the nuclear factor κB (NF-κB) subunit p65 in aortic endothelial cells both in vitro and in nondiabetic mice, which cause increased p65 gene expression. Both the epigenetic changes and the gene expression changes persist for at least 6 d of subsequent normal glycemia, as do NF-κB–induced increases in monocyte chemoattractant protein 1 and vascular cell adhesion molecule 1 expression. Hyperglycemia-induced epigenetic changes and increased p65 expression are prevented by reducing mitochondrial superoxide production or superoxide-induced α-oxoaldehydes. These results highlight the dramatic and long-lasting effects that short-term hyperglycemic spikes can have on vascular cells and suggest that transient spikes of hyperglycemia may be an HbA1c–independent risk factor for diabetic complications.

Interaction of a gene-specific transcription factor with the adenovirus major late promoter upstream of the TATA box region

A gene-specific transcription factor, called USF, has been partially purified from HeLa cell nuclear extracts. Addition of USF results in a 10 to 20 fold increase in transcription from the adenovirus major late promoter in an in vitro system reconstituted with transcription factors TFIIB, TFIID, TFIIE, and RNA polymerase II. Binding of USF to the promoter inhibits DNAase I cleavages over a 20 base pair region just upstream of the -45 to +35 region shown previously to interact with TFIID. More discriminating footprint analyses using methidiumpropyl-EDTA-Fe(II) as the cleaving agent indicate that USF interacts primarily with the small palindromic DNA sequence GGCCACGTGACC located between positions -63 and -52 of the major late promoter, while TFIID interacts primarily with a 10 base pair DNA region centered on the consensus TATA sequence. Dissociation rate measurements indicate a cooperative interaction between USF and TFIID when simultaneously bound to the promoter DNA.

WDR5 associates with histone H3 methylated at K4 and is essential for H3 K4 methylation and vertebrate development.

Histone H3 lysine 4 (K4) methylation has been linked to the transcriptional activation in a variety of eukaryotic species. Here we show that a common component of MLL1, MLL2, and hSet1 H3 K4 methyltransferase complexes, the WD40-repeat protein WDR5, directly associates with histone H3 di- and trimethylated at K4 and with H3-K4-dimethylated nucleosomes. WDR5 is required for binding of the methyltransferase complex to the K4-dimethylated H3 tail as well as for global H3 K4 trimethylation and HOX gene activation in human cells. WDR5 is essential for vertebrate development, in that WDR5-depleted X. laevis tadpoles exhibit a variety of developmental defects and abnormal spatial Hox gene expression. Our results are the first demonstration that a WD40-repeat protein acts as a module for recognition of a specific histone modification and suggest a mechanism for reading and writing an epigenetic mark for gene activation.

Synergistic activation of transcription by CBP and p53.

The tumour suppressor p53 is a transcriptional regulator whose ability to inhibit cell growth is dependent upon its transactivation function. Here we demonstrate that the transcription factor CBP, which is also implicated in cell proliferation and differentiation, acts as a p53 coactivator and potentiates its transcriptional activity. The amino-terminal activation domain of p53 interacts with the carboxy-terminal portion of the CBP protein both in vitro and in vivo. In transfected SaoS-2 cells, CBP potentiates activation of the mdm-2 gene by p53 and, reciprocally, p53 potentiates activation of a Gal4-responsive target gene by a Gal4(1–147)–CBP(1678–2441) fusion protein. A double point mutation that destroys the transactivation function of p53 also abolishes its binding to CBP and its synergistic function with CBP. The ability of p53 to interact physically and functionally with a co-activator (CBP) that has histone acetyltransferase activity, and with components (TAFs), of the general transcription machinery indicates that it may have different functions in a multistep activation pathway.

Physical association and coordinate function of the H3 K4 methyltransferase MLL1 and the H4 K16 acetyltransferase MOF

A stable complex containing MLL1 and MOF has been immunoaffinity purified from a human cell line that stably expresses an epitope-tagged WDR5 subunit. Stable interactions between MLL1 and MOF were confirmed by reciprocal immunoprecipitation, cosedimentation, and cotransfection analyses, and interaction sites were mapped to MLL1 C-terminal and MOF zinc finger domains. The purified complex has a robust MLL1-mediated histone methyltransferase activity that can effect mono-, di-, and trimethylation of H3 K4 and a MOF-mediated histone acetyltransferase activity that is specific for H4 K16. Importantly, both activities are required for optimal transcription activation on a chromatin template in vitro and on an endogenous MLL1 target gene, Hox a9, in vivo. These results indicate an activator-based mechanism for joint MLL1 and MOF recruitment and targeted methylation and acetylation and provide a molecular explanation for the closely correlated distribution of H3 K4 methylation and H4 K16 acetylation on active genes.

Regulation of MLL1 H3K4 methyltransferase activity by its core components

Histone H3 Lys4 (H3K4) methylation is a prevalent mark associated with transcription activation. A common feature of several H3K4 methyltransferase complexes is the presence of three structural components (RbBP5, Ash2L and WDR5) and a catalytic subunit containing a SET domain. Here we report the first biochemical reconstitution of a functional four-component mixed-lineage leukemia protein-1 (MLL1) core complex. This reconstitution, combined with in vivo assays, allows direct analysis of the contribution of each component to MLL1 enzymatic activity and their roles in transcriptional regulation. Moreover, taking clues from a crystal structure analysis, we demonstrate that WDR5 mediates interactions of the MLL1 catalytic unit both with the common structural platform and with the histone substrate. Mechanistic insights gained from this study can be generalized to the whole family of SET1-like histone methyltransferases in mammals.

The metazoan Mediator co-activator complex as an integrative hub for transcriptional regulation

The Mediator is an evolutionarily conserved, multiprotein complex that is a key regulator of protein-coding genes. In metazoan cells, multiple pathways that are responsible for homeostasis, cell growth and differentiation converge on the Mediator through transcriptional activators and repressors that target one or more of the almost 30 subunits of this complex. Besides interacting directly with RNA polymerase II, Mediator has multiple functions and can interact with and coordinate the action of numerous other co-activators and co-repressors, including those acting at the level of chromatin. These interactions ultimately allow the Mediator to deliver outputs that range from maximal activation of genes to modulation of basal transcription to long-term epigenetic silencing.

S Phase Activation of the Histone H2B Promoter by OCA-S, a Coactivator Complex that Contains GAPDH as a Key Component

We have isolated and functionally characterized a multicomponent Oct-1 coactivator, OCA-S which is essential for S phase-dependent histone H2B transcription. The p38 component of OCA-S binds directly to Oct-1, exhibits potent transactivation potential, is selectively recruited to the H2B promoter in S phase, and is essential for S phase-specific H2B transcription in vivo and in vitro. Surprisingly, p38 represents a nuclear form of glyceraldehyde-3-phosphate dehydrogenase, and binding to Oct-1, as well as OCA-S function, is stimulated by NAD(+) but inhibited by NADH. OCA-S also interacts with NPAT, a cyclin E/cdk2 substrate that is broadly involved in histone gene transcription. These studies thus link the H2B transcriptional machinery to cell cycle regulators, and possibly to cellular metabolic state (redox status), and set the stage for studies of the underlying mechanisms and the basis for coordinated histone gene expression and coupling to DNA replication.