Aidong Han

FOXP3 Controls Regulatory T Cell Function through Cooperation with NFAT

Antigen stimulation of immune cells activates the transcription factor NFAT, a key regulator of T cell activation and anergy. NFAT forms cooperative complexes with the AP-1 family of transcription factors and regulates T cell activation-associated genes. Here we show that regulatory T cell (Treg) function is mediated by an analogous cooperative complex of NFAT with the forkhead transcription factor FOXP3, a lineage specification factor for Tregs. The crystal structure of an NFAT:FOXP2:DNA complex reveals an extensive protein-protein interaction interface between NFAT and FOXP2. Structure-guided mutations of FOXP3, predicted to progressively disrupt its interaction with NFAT, interfere in a graded manner with the ability of FOXP3 to repress expression of the cytokine IL2, upregulate expression of the Treg markers CTLA4 and CD25, and confer suppressor function in a murine model of autoimmune diabetes. Thus by switching transcriptional partners, NFAT converts the acute T cell activation program into the suppressor program of Tregs.

Mechanistic Insights Revealed by the Crystal Structure of a Histidine Kinase with Signal Transducer and Sensor Domains

A crystal structure reveals an elegant mechanistic switch whereby helical bending and catalytic domain rotation allow self-activation of a histidine kinase during a bacterial stress response.

Sequence-specific recruitment of transcriptional co-repressor Cabin1 by myocyte enhancer factor-2

The myocyte enhancer factor-2 (MEF2) family of transcription factors has important roles in the development and function of T cells, neuronal cells and muscle cells. MEF2 is capable of repressing or activating transcription by association with a variety of co-repressors or co-activators in a calcium-dependent manner. Transcriptional repression by MEF2 has attracted particular attention because of its potential role in hypertrophic responses of cardiomyocytes. Several MEF2 co-repressors, such as Cabin1/Cain and class II histone deacetylases (HDACs), have been identified. However, the molecular mechanism of their recruitment to specific promoters by MEF2 remains largely unknown. Here we report a crystal structure of the MADS-box/MEF2S domain of human MEF2B bound to a motif of the transcriptional co-repressor Cabin1 and DNA at 2.2 Å resolution. The crystal structure reveals a stably folded MEF2S domain on the surface of the MADS box. Cabin1 adopts an amphipathic α-helix to bind a hydrophobic groove on the MEF2S domain, forming a triple-helical interaction. Our studies of the ternary Cabin1/MEF2/DNA complex show a general mechanism by which MEF2 recruits transcriptional co-repressor Cabin1 and class II HDACs to specific DNA sites.

Crystal structure of a conserved N-terminal domain of histone deacetylase 4 reveals functional insights into glutamine-rich domains

Glutamine-rich sequences exist in a wide range of proteins across multiple species. A subset of glutamine-rich sequences has been shown to form amyloid fibers implicated in human diseases. The physiological functions of these sequence motifs are not well understood, partly because of the lack of structural information. Here we have determined a high-resolution structure of a glutamine-rich domain from human histone deacetylase 4 (HDAC4) by x-ray crystallography. The glutamine-rich domain of HDAC4 (19 glutamines of 68 residues) folds into a straight α-helix that assembles as a tetramer. In contrast to most coiled coil proteins, the HDAC4 tetramer lacks regularly arranged apolar residues and an extended hydrophobic core. Instead, the protein interfaces consist of multiple hydrophobic patches interspersed with polar interaction networks, wherein clusters of glutamines engage in extensive intra- and interhelical interactions. In solution, the HDAC4 tetramer undergoes rapid equilibrium with monomer and intermediate species. Structure-guided mutations that expand or disrupt hydrophobic patches drive the equilibrium toward the tetramer or monomer, respectively. We propose that a general role of glutamine-rich motifs be to mediate protein–protein interactions characteristic of a large component of polar interaction networks that may facilitate reversible assembly and disassembly of protein complexes.

Signal-induced Brd4 release from chromatin is essential for its role transition from chromatin targeting to transcriptional regulation

Bromodomain-containing protein Brd4 is shown to persistently associate with chromosomes during mitosis for transmitting epigenetic memory across cell divisions. During interphase, Brd4 also plays a key role in regulating the transcription of signal-inducible genes by recruiting positive transcription elongation factor b (P-TEFb) to promoters. How the chromatin-bound Brd4 transits into a transcriptional regulation mode in response to stimulation, however, is largely unknown. Here, by analyzing the dynamics of Brd4 during ultraviolet or hexamethylene bisacetamide treatment, we show that the signal-induced release of chromatin-bound Brd4 is essential for its functional transition. In untreated cells, almost all Brd4 is observed in association with interphase chromatin. Upon treatment, Brd4 is released from chromatin, mostly due to signal-triggered deacetylation of nucleosomal histone H4 at acetylated-lysine 5/8 (H4K5ac/K8ac). Through selective association with the transcriptional active form of P-TEFb that has been liberated from the inactive multi-subunit complex in response to treatment, the released Brd4 mediates the recruitment of this active P-TEFb to promoter, which enhances transcription at the stage of elongation. Thus, through signal-induced release from chromatin and selective association with the active form of P-TEFb, the chromatin-bound Brd4 switches its role to mediate the recruitment of P-TEFb for regulating the transcriptional elongation of signal-inducible genes.

Structure of p300 bound to MEF2 on DNA reveals a mechanism of enhanceosome assembly

Transcription co-activators CBP and p300 are recruited by sequence-specific transcription factors to specific genomic loci to control gene expression. A highly conserved domain in CBP/p300, the TAZ2 domain, mediates direct interaction with a variety of transcription factors including the myocyte enhancer factor 2 (MEF2). Here we report the crystal structure of a ternary complex of the p300 TAZ2 domain bound to MEF2 on DNA at 2.2Å resolution. The structure reveals three MEF2:DNA complexes binding to different sites of the TAZ2 domain. Using structure-guided mutations and a mammalian two-hybrid assay, we show that all three interfaces contribute to the binding of MEF2 to p300, suggesting that p300 may use one of the three interfaces to interact with MEF2 in different cellular contexts and that one p300 can bind three MEF2:DNA complexes simultaneously. These studies, together with previously characterized TAZ2 complexes bound to different transcription factors, demonstrate the potency and versatility of TAZ2 in protein–protein interactions. Our results also support a model wherein p300 promotes the assembly of a higher-order enhanceosome by simultaneous interactions with multiple DNA-bound transcription factors.

Structural Basis of HIV-1 Activation by NF-κB—A Higher-Order Complex of p50:RelA Bound to the HIV-1 LTR

The activation and latency of human immunodeficiency virus type 1 (HIV-1) are tightly controlled by the transcriptional activity of its long terminal repeat (LTR) region. The LTR is regulated by viral proteins as well as host factors, including the nuclear factor kappaB (NF-kappaB) that becomes activated in virus-infected cells. The two tandem NF-kappaB sites of the LTR are among the most highly conserved sequence elements of the HIV-1 genome. Puzzlingly, these sites are arranged in a manner that seems to preclude simultaneous binding of both sites by NF-kappaB, although previous biochemical work suggests otherwise. Here, we have determined the crystal structure of p50:RelA bound to the tandem kappaB element of the HIV-1 LTR as a dimeric dimer, providing direct structural evidence that NF-kappaB can occupy both sites simultaneously. The two p50:RelA dimers bind the adjacent kappaB sites and interact through a protein contact that is accommodated by DNA bending. The two dimers clamp DNA from opposite faces of the double helix and form a topological trap of the bound DNA. Consistent with these structural features, our biochemical analyses indicate that p50:RelA binds the HIV-1 LTR tandem kappaB sites with an apparent anti-cooperativity but enhanced kinetic stability. The slow on and off rates we observe may be relevant to viral latency because viral activation requires sustained NF-kappaB activation. Furthermore, our work demonstrates that the specific arrangement of the two kappaB sites on the HIV-1 LTR can modulate the assembly kinetics of the higher-order NF-kappaB complex on the viral promoter. This phenomenon is unlikely restricted to the HIV-1 LTR but probably represents a general mechanism for the function of composite DNA elements in transcription.

Inhibition of the function of class IIa HDACs by blocking their interaction with MEF2

Enzymes that modify the epigenetic status of cells provide attractive targets for therapy in various diseases. The therapeutic development of epigenetic modulators, however, has been largely limited to direct targeting of catalytic active site conserved across multiple members of an enzyme family, which complicates mechanistic studies and drug development. Class IIa histone deacetylases (HDACs) are a group of epigenetic enzymes that depends on interaction with Myocyte Enhancer Factor-2 (MEF2) for their recruitment to specific genomic loci. Targeting this interaction presents an alternative approach to inhibiting this class of HDACs. We have used structural and functional approaches to identify and characterize a group of small molecules that indirectly target class IIa HDACs by blocking their interaction with MEF2 on DNA.Weused X-ray crystallography and 19F NMRto show that these compounds directly bind to MEF2. We have also shown that the small molecules blocked the recruitment of class IIa HDACs to MEF2-targeted genes to enhance the expression of those targets. These compounds can be used as tools to study MEF2 and class IIa HDACs in vivo and as leads for drug development.

Structure of the MADS-box/MEF2 Domain of MEF2A Bound to DNA and Its Implication for Myocardin Recruitment

Myocyte enhancer factor 2 (MEF2) regulates specific gene expression in diverse developmental programs and adaptive responses. MEF2 recognizes DNA and interacts with transcription cofactors through a highly conserved N-terminal domain referred to as the MADS-box/MEF2 domain. Here we present the crystal structure of the MADS-box/MEF2 domain of MEF2A bound to DNA. In contrast to previous structural studies showing that the MEF2 domain of MEF2A is partially unstructured, the present study reveals that the MEF2 domain participates with the MADS-box in both dimerization and DNA binding as a single domain. The sequence divergence at and immediately following the C-terminal end of the MEF2 domain may allow different MEF2 dimers to recognize different DNA sequences in the flanking regions. The current structure also suggests that the ligand-binding pocket previously observed in the Cabin1-MEF2B-DNA complex and the HDAC9 (histone deacetylase 9)-MEF2B-DNA complex is not induced by cofactor binding but rather preformed by intrinsic folding. However, the structure of the ligand-binding pocket does undergo subtle but significant conformational changes upon cofactor binding. On the basis of these observations, we generated a homology model of MEF2 bound to a myocardin family protein, MASTR, that acts as a potent coactivator of MEF2-dependent gene expression. The model shows excellent shape and chemical complementarity at the binding interface and is consistent with existing mutagenesis data. The apo structure presented here can also serve as a target for virtual screening and soaking studies of small molecules that can modulate the function of MEF2 as research tools and therapeutic leads.

Histone Cross-talk Connects Protein Phosphatase 1α (PP1α) and Histone Deacetylase (HDAC) Pathways to Regulate the Functional Transition of Bromodomain-containing 4 (BRD4) for Inducible Gene Expression

Background: Transcription elongation is a rate-limiting step for inducible gene expression. BRD4 must be released from chromatin to regulate transcription elongation. Results: Protein phosphatase 1α (PP1α) and histone deacetylases (HDAC) signaling pathways are required for this process. Conclusion: Histone cross-talk in trans between H3S10ph and H4K5ac/K8ac connects PP1α and HDACs signaling pathways to control functional transition of BRD4. Significance: BRD4 is regulated epigenetically for controlling stress-induced gene expression. Transcription elongation has been recognized as a rate-limiting step for the expression of signal-inducible genes. Through recruitment of positive transcription elongation factor P-TEFb, the bromodomain-containing protein BRD4 plays critical roles in regulating the transcription elongation of a vast array of inducible genes that are important for multiple cellular processes. The diverse biological roles of BRD4 have been proposed to rely on its functional transition between chromatin targeting and transcription regulation. The signaling pathways and the molecular mechanism for regulating this transition process, however, are largely unknown. Here, we report a novel role of phosphorylated Ser10 of histone H3 (H3S10ph) in governing the functional transition of BRD4. We identified that the acetylated lysines 5 and 8 of nucleosomal histone H4 (H4K5ac/K8ac) is the BRD4 binding site, and the protein phosphatase PP1α and class I histone deacetylase (HDAC1/2/3) signaling pathways are essential for the stress-induced BRD4 release from chromatin. In the unstressed state, phosphorylated H3S10 prevents the deacetylation of nucleosomal H4K5ac/K8ac by HDAC1/2/3, thereby locking up the majority of BRD4 onto chromatin. Upon stress, PP1α-mediated dephosphorylation of H3S10ph allows the deacetylation of nucleosomal H4K5ac/K8ac by HDAC1/2/3, thereby leading to the release of chromatin-bound BRD4 for subsequent recruitment of P-TEFb to enhance the expression of inducible genes. Therefore, our study revealed a novel mechanism that the histone cross-talk between H3S10ph and H4K5ac/K8ac connects PP1α and HDACs to govern the functional transition of BRD4. Combined with previous studies on the regulation of P-TEFb activation, the intricate signaling network for the tight control of transcription elongation is established.