Anja Schuetz

Human HDAC7 harbors a class IIa histone deacetylase-specific zinc binding motif and cryptic deacetylase activity.

Histone deacetylases (HDACs) are protein deacetylases that play a role in repression of gene transcription and are emerging targets in cancer therapy. Here, we characterize the structure and enzymatic activity of the catalytic domain of human HDAC7 (cdHDAC7). Although HDAC7 normally exists as part of a multiprotein complex, we show that cdHDAC7 has a low level of deacetylase activity which can be inhibited by known HDAC inhibitors. The crystal structures of human cdHDAC7 and its complexes with two hydroxamate inhibitors are the first structures of the catalytic domain of class IIa HDACs and demonstrate significant differences with previously reported class I and class IIb-like HDAC structures. We show that cdHDAC7 has an additional class IIa HDAC-specific zinc binding motif adjacent to the active site which is likely to participate in substrate recognition and protein-protein interaction and may provide a site for modulation of activity. Furthermore, a different active site topology results in modified catalytic properties and in an enlarged active site pocket. Our studies provide mechanistic insights into class IIa HDACs and facilitate the design of specific modulators.

Structural basis for molecular recognition and presentation of histone H3 by WDR5.

Histone methylation at specific lysine residues brings about various downstream events that are mediated by different effector proteins. The WD40 domain of WDR5 represents a new class of histone methyl‐lysine recognition domains that is important for recruiting H3K4 methyltransferases to K4‐dimethylated histone H3 tail as well as for global and gene‐specific K4 trimethylation. Here we report the crystal structures of full‐length WDR5, WDR5Δ23 and its complexes with unmodified, mono‐, di‐ and trimethylated histone H3K4 peptides. The structures reveal that WDR5 is able to bind all of these histone H3 peptides, but only H3K4me2 peptide forms extra interactions with WDR5 by use of both water‐mediated hydrogen bonding and the altered hydrophilicity of the modified lysine 4. We propose a mechanism for the involvement of WDR5 in binding and presenting histone H3K4 for further methylation as a component of MLL complexes.

The Structural Basis of Gas-Responsive Transcription by the Human Nuclear Hormone Receptor REV-ERBβ

Heme is a ligand for the human nuclear receptors (NR) REV-ERBα and REV-ERBβ, which are transcriptional repressors that play important roles in circadian rhythm, lipid and glucose metabolism, and diseases such as diabetes, atherosclerosis, inflammation, and cancer. Here we show that transcription repression mediated by heme-bound REV-ERBs is reversed by the addition of nitric oxide (NO), and that the heme and NO effects are mediated by the C-terminal ligand-binding domain (LBD). A 1.9 Å crystal structure of the REV-ERBβ LBD, in complex with the oxidized Fe(III) form of heme, shows that heme binds in a prototypical NR ligand-binding pocket, where the heme iron is coordinately bound by histidine 568 and cysteine 384. Under reducing conditions, spectroscopic studies of the heme-REV-ERBβ complex reveal that the Fe(II) form of the LBD transitions between penta-coordinated and hexa-coordinated structural states, neither of which possess the Cys384 bond observed in the oxidized state. In addition, the Fe(II) LBD is also able to bind either NO or CO, revealing a total of at least six structural states of the protein. The binding of known co-repressors is shown to be highly dependent upon these various liganded states. REV-ERBs are thus highly dynamic receptors that are responsive not only to heme, but also to redox and gas. Taken together, these findings suggest new mechanisms for the systemic coordination of molecular clocks and metabolism. They also raise the possibility for gas-based therapies for the many disorders associated with REV-ERB biological functions.

The Structure of the Klf4 DNA-Binding Domain Links to Self-Renewal and Macrophage Differentiation.

Krueppel-like factor 4 (Klf4) belongs to the Sp/Klf family of zinc-finger transcription factors and is indispensable for terminal maturation of epithelial tissues. Furthermore, it is part of a small set of proteins that are used to generate pluripotent embryonic stem cells from differentiated tissues. Herein, we describe that a Klf4 zinc-finger domain mutant induces self-renewal and block of maturation, while wild-type Klf4 induces terminal macrophage differentiation. Moreover, we present the crystal structure of the zinc-finger domain of Klf4 bound to its target DNA, revealing that primarily the two C-terminal zinc-finger motifs are required for site specificity. Lack of those two zinc fingers leads to deficiency of Klf4 to induce macrophage differentiation. The first zinc finger, on the other hand, inhibits the otherwise cryptic self-renewal and block of differentiation activity of Klf4. Our data show that impairing the DNA binding could potentially contribute to a monocytic leukemia.

RC3H1 post-transcriptionally regulates A20 mRNA and modulates the activity of the IKK/NF-κB pathway.

The RNA-binding protein RC3H1 (also known as ROQUIN) promotes TNFα mRNA decay via a 3′UTR constitutive decay element (CDE). Here we applied PAR-CLIP to human RC3H1 to identify ∼3,800 mRNA targets with >16,000 binding sites. A large number of sites are distinct from the consensus CDE and revealed a structure-sequence motif with U-rich sequences embedded in hairpins. RC3H1 binds preferentially short-lived and DNA damage-induced mRNAs, indicating a role of this RNA-binding protein in the post-transcriptional regulation of the DNA damage response. Intriguingly, RC3H1 affects expression of the NF-κB pathway regulators such as IκBα and A20. RC3H1 uses ROQ and Zn-finger domains to contact a binding site in the A20 3′UTR, demonstrating a not yet recognized mode of RC3H1 binding. Knockdown of RC3H1 resulted in increased A20 protein expression, thereby interfering with IκB kinase and NF-κB activities, demonstrating that RC3H1 can modulate the activity of the IKK/NF-κB pathway.

The SNF2-like helicase HELLS mediates E2F3-dependent transcription and cellular transformation

The activating E2F‐transcription factors are best known for their dependence on the Retinoblastoma protein and their role in cellular proliferation. E2F3 is uniquely amplified in specific human tumours where its expression is inversely correlated with the survival of patients. Here, E2F3B interaction partners were identified by mass spectrometric analysis. We show that the SNF2‐like helicase HELLS interacts with E2F3A in vivo and cooperates with its oncogenic functions. Depletion of HELLS severely perturbs the induction of E2F‐target genes, hinders cell‐cycle re‐entry and growth. Using chromatin immmunoprecipitation coupled to sequencing, we identified genome‐wide targets of HELLS and E2F3A/B. HELLS binds promoters of active genes, including the trithorax‐related MLL1, and co‐regulates E2F3‐dependent genes. Strikingly, just as E2F3, HELLS is overexpressed in human tumours including prostate cancer, indicating that either factor may contribute to the malignant progression of tumours. Our work reveals that HELLS is important for E2F3 in tumour cell proliferation.

Crystal structure of a binary complex between human GCN5 histone acetyltransferase domain and acetyl coenzyme A

Becauseof this, enzymes catalyzing histone acetylation, the his-tone acetyltransferases (HATs, EC 2.3.1.48), have a majorrole in the cell fate, proliferation, and differentiation.The histone acetyltransferase GCN5 acts as a supervi-sor in the normal cell cycle progression having compre-hensive control over expressions of these cell cycle-related genes, as well as apoptosis-related genes, viaalterations in the chromatin structure, mimicked bychanging acetylation status of core histones, which sur-round these genes.

Molecular Insights into Arrhythmogenic Right Ventricular Cardiomyopathy Caused by Plakophilin-2 Missense Mutations

Background—Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiac disorder mainly caused by dominant mutations in several components of the cardiac desmosome including plakophilin-2 (PKP2), the most prevalent disease gene. Little is known about the underlying genetic and molecular mechanisms of missense mutations located in the armadillo (ARM) domains of PKP2, as well as their consequences on human cardiac pathology. Methods and Results—We focused on in vivo and in vitro studies of the PKP2 founder mutation c.2386T>C (p.C796R), and demonstrated in cardiac tissue from 2 related mutation carriers a patchy expression pattern ranging from unchanged to totally absent immunoreactive signals of PKP2 and other desmosomal proteins. In vitro expression analysis of mutant PKP2 in cardiac derived HL-1 cells revealed unstable proteins that fail to interact with desmoplakin and are targeted by degradation involving calpain proteases. Bacterial expression, crystallization, and structural modeling of mutated proteins impacting different ARM domains and helices of PKP2 confirmed their instability and degradation, resulting in the same remaining protein fragment that was crystallized and used to model the entire ARM domain of PKP2. Conclusions—The p.C796R and other ARVC-related PKP2 mutations indicate loss of function effects by intrinsic instability and calpain proteases mediated degradation in in vitro model systems, suggesting haploinsufficiency as the most likely cause for the genesis of dominant ARVC due to mutations in PKP2.

Repression of Transcriptional Activity of C/EBPα by E2F-Dimerization Partner Complexes

ABSTRACT The transcription factor CCAAT/enhancer-binding protein α (C/EBPα) coordinates proliferation arrest and the differentiation of myeloid progenitors, adipocytes, hepatocytes, keratinocytes, and cells of the lung and placenta. C/EBPα transactivates lineage-specific differentiation genes and inhibits proliferation by repressing E2F-regulated genes. The myeloproliferative C/EBPα BRM2 mutant serves as a paradigm for recurrent human C-terminal bZIP C/EBPα mutations that are involved in acute myeloid leukemogenesis. BRM2 fails to repress E2F and to induce adipogenesis and granulopoiesis. The data presented here show that, independently of pocket proteins, C/EBPα interacts with the dimerization partner (DP) of E2F and that C/EBPα-E2F/DP interaction prevents both binding of C/EBPα to its cognate sites on DNA and transactivation of C/EBP target genes. The BRM2 mutant, in addition, exhibits enhanced interaction with E2F-DP and reduced affinity toward DNA and yet retains transactivation potential and differentiation competence that becomes exposed when E2F/DP levels are low. Our data suggest a tripartite balance between C/EBPα, E2F/DP, and pocket proteins in the control of proliferation, differentiation, and tumorigenesis.

Crystal structure of the tricellulin C-terminal coiled-coil domain reveals a unique mode of dimerization.

Tricellulin is a tight junction protein localized to tricellular contacts in many epithelial tissues, where it is required for full barrier control. Here, we present crystal structures of the tricellulin C‐terminal coiled‐coil domain, revealing a potential dimeric arrangement. By combining structural, biochemical, functional, and mutation analyses, we gain insight into the mode of tricellulin oligomerization and suggest a model where dimerization of its cytoplasmic C‐terminus may play an auxiliary role in stabilizing homophilic and potentially also heterophilic cis‐interactions within tight junctions.