Evolutionary Conservation of Structural and Functional Coupling between the BRM AT-Hook and Bromodomain

The BAF chromatin remodeling complex is critical for genome regulation. The central ATPase of BAF is either BRM or BRG1, both of which contain a C-terminal bromodomain, known to associate with acetylated lysines. We have recently demonstrated that in addition to acetyl-lysine binding, the BRG1/BRM bromodomain can associate with DNA through a lysine/arginine rich patch that is adjacent to the acetyl-lysine binding pocket. Flanking the bromodomain is an AT-hook separated by a short, proline-rich linker. We previously found that the AT-hook and bromodomain can associate with DNA in a multivalent manner. Here, we investigate the conservation of this composite module and find that the AT-hook, linker, and lysine/arginine rich bromodomain patch are ancient, conserved over ~1 billion years. We utilize extensive mutagenesis, NMR spectroscopy, and fluorescence anisotropy to dissect the contribution of each of these conserved elements in association of this module with DNA. Our results reveal a structural and functional coupling of the AT-hook and bromodomain mediated by the linker. The lysine/arginine rich patch on the bromodomain and the conserved elements of the AT-hook are critical for robust affinity for DNA, while the conserved elements of the linker are dispensable for overall DNA affinity but critical for maintaining the relative conformation of the AT-hook and bromodomain in binding to DNA. This supports that the coupled action of the AT-hook and bromodomain are important for BAF activity.     

Regulation of CSF1 promoter by the SWI/SNF-like BAF complex

The mammalian BAF complex regulates gene expression by modifying chromatin structure. In this report, we identify 80 genes activated and 2 genes repressed by the BAF complex in SW-13 cells. We find that prior binding of NFI/CTF to the NFI/CTF binding site in CSF1 promoter is required for the recruitment of the BAF complex and the BAF-dependent activation of the promoter. Furthermore, the activation of the CSF1 promoter requires Z-DNA-forming sequences that are converted to Z-DNA structure upon activation by the BAF complex. The BAF complex facilitates Z-DNA formation in a nucleosomal template in vitro. We propose a model in which the BAF complex promotes Z-DNA formation which, in turn, stabilizes the open chromatin structure at the CSF1 promoter.     

Barrier-to-autointegration factor: major roles in chromatin decondensation and nuclear assembly

Barrier-to-autointegration factor (BAF) is a DNA-bridging protein, highly conserved in metazoans. BAF binds directly to LEM (LAP2, emerin, MAN1) domain nuclear membrane proteins, including LAP2 and emerin. We used site-directed mutagenesis and biochemical analysis to map functionally important residues in human BAF, including those required for direct binding to DNA or emerin. We also tested wild-type BAF and 25 point mutants for their effects on nuclear assembly in Xenopus egg extracts, which contain approximately 12 microM endogenous BAF dimers. Exogenous BAF caused two distinct effects: at low added concentrations, wild-type BAF enhanced chromatin decondensation and nuclear growth; at higher added concentrations, wild-type BAF completely blocked chromatin decondensation and nuclear growth. Mutants fell into four classes, including one that defines a novel functional surface on the BAF dimer. Our results suggest that BAF, unregulated, potently compresses chromatin structure, and that BAF interactions with both DNA and LEM proteins are critical for membrane recruitment and chromatin decondensation during nuclear assembly.     

Nucleophosmin mutations in acute myeloid leukemia: A tale of protein unfolding and mislocalization

Nucleophosmin (NPM1) is an abundant, ubiquitously expressed protein mainly localized at nucleoli but continuously shuttling between nucleus and cytoplasm. NPM1 plays a role in several cellular functions, including ribosome biogenesis and export, centrosome duplication, chromatin remodeling, DNA repair, and response to stress stimuli. Much of the interest in this protein arises from its relevance in human malignancies. NPM1 is frequently overexpressed in solid tumors and is the target of several chromosomal translocations in hematologic neoplasms. Notably, NPM1 has been characterized as the most frequently mutated gene in acute myeloid leukemia (AML). Mutations alter the C‐terminal DNA‐binding domain of the protein and result in its aberrant nuclear export and stable cytosolic localization. In this review, we focus on the leukemia‐associated NPM1 C‐terminal domain and describe its structure, function, and the effect exerted by leukemic mutations. Finally, we discuss the possibility to target NPM1 for the treatment of cancer and, in particular, of AML patients with mutated NPM1 gene.     

Structural basis of DNA bridging by barrier-to-autointegration factor

Barrier-to-autointegration factor (BAF) is a host cell protein that plays a crucial role in retroviral integration. Preintegration complexes (PICs) stripped of BAF lose their normal integration activity, which can be restored by incubation with purified BAF. BAF bridges double-stranded DNA both intra- and intermolecularly in a non-sequence-specific manner, leading to the formation of a nucleoprotein network. BAF also binds to the nuclear protein lamina-associated polypeptide 2 (LAP2), and is localized with chromatin during interphase and mitosis. The crystal structure of homodimeric human BAF has been determined to 1.9 A resolution. The fold of the BAF monomer resembles that of the second domain of RuvA. This comparison revealed the presence of the helix-hairpin-helix (HhH) nonspecific DNA binding motif within BAF. A novel feature of BAF's HhH motif is the occupation of the metal binding site by the epsilon-amino group of Lys 6, providing an alternative means of sequestering positive charge. Mutational analysis corroborates the HhH motif's prominent role in DNA binding and argues against a previously proposed helix-turn-helix (HTH) binding site located in another region of the monomer. A model of BAF bridging DNA via the HhH motif is proposed.     

Two Chromatin Remodeling Activities Cooperate during Activation of Hormone Responsive Promoters

Steroid hormones regulate gene expression by interaction of their receptors with hormone responsive elements (HREs) and recruitment of kinases, chromatin remodeling complexes, and coregulators to their target promoters. Here we show that in breast cancer cells the BAF, but not the closely related PBAF complex, is required for progesterone induction of several target genes including MMTV, where it catalyzes localized displacement of histones H2A and H2B and subsequent NF1 binding. PCAF is also needed for induction of progesterone target genes and acetylates histone H3 at K14, an epigenetic mark that interacts with the BAF subunits by anchoring the complex to chromatin. In the absence of PCAF, full loading of target promoters with hormone receptors and BAF is precluded, and induction is compromised. Thus, activation of hormone-responsive promoters requires cooperation of at least two chromatin remodeling activities, BAF and PCAF.     

LAP2 binds to BAF.DNA complexes: requirement for the LEM domain and modulation by variable regions

LAP2 belongs to a family of nuclear membrane proteins sharing a 43 residue LEM domain. All LAP2 isoforms have the same N-terminal 'constant' region (LAP2-c), which includes the LEM domain, plus a C-terminal 'variable' region. LAP2-c polypeptide inhibits nuclear assembly in Xenopus extracts, and binds in vitro to barrier-to-autointegration factor (BAF), a DNA-bridging protein. We tested 17 Xenopus LAP2-c mutants for nuclear assembly inhibition, and binding to BAF and BAF small middle dotDNA complexes. LEM domain mutations disrupted all activities tested. Some mutations outside the LEM domain had no effect on binding to BAF, but disrupted activity in Xenopus extracts, suggesting that LAP2-c has an additional unknown function required to inhibit nuclear assembly. Mutagenesis results suggest that BAF changes conformation when complexed with DNA. The binding affinity of LAP2 was higher for BAF small middle dotDNA complexes than for BAF, suggesting that these interactions are physiologically relevant. Nucleoplasmic domains of XENOPUS: LAP2 isoforms varied 9-fold in their affinities for BAF, but all isoforms supershifted BAF small middle dotDNA complexes. We propose that the LEM domain is a core BAF-binding domain that can be modulated by the variable regions of LAP2 isoforms.     

抑癌基因ARID1A在肿瘤中的研究进展

染色质重塑复合物相关基因在癌症中频繁突变,这种现象逐渐引起研究者的重视。然而,染色质重塑活动如何引起癌症发生,对此机理研究甚少。ARID1A是SWl／SNF（BRG1相关因子）染色质重塑复合物中的一个亚基,具有DNA结合活性,可以与富含AT的DNA序列特异性结合。近来基因组测序发现,ARID1A在卵巢癌、肝癌、胃癌、乳腺癌等肿瘤中频繁发生突变,这些突变导致ARID1A在肿瘤中表达降低,表明ARID1A是个潜在的抑癌基因。该文将针对ARID1A在各种癌症中的缺失及失活机制、ARID1A的生物学功能和潜在抑癌机理以及与,临床预后之间关系等方面做一综述,以期为肿瘤诊断、治疗提供新思路。     

Swi1 Associates with Chromatin through the DDT Domain and Recruits Swi3 to Preserve Genomic Integrity

Swi1 and Swi3 form the replication fork protection complex and play critical roles in proper activation of the replication checkpoint and stabilization of replication forks in the fission yeast Schizosaccharomyces pombe. However, the mechanisms by which the Swi1-Swi3 complex regulates these processes are not well understood. Here, we report functional analyses of the Swi1-Swi3 complex in fission yeast. Swi1 possesses the DDT domain, a putative DNA binding domain found in a variety of chromatin remodeling factors. Consistently, the DDT domain-containing region of Swi1 interacts with DNA in vitro, and mutations in the DDT domain eliminate the association of Swi1 with chromatin in S. pombe cells. DDT domain mutations also render cells highly sensitive to S-phase stressing agents and induce strong accumulation of Rad22-DNA repair foci, indicating that the DDT domain is involved in the activity of the Swi1-Swi3 complex. Interestingly, DDT domain mutations also abolish Swi1's ability to interact with Swi3 in cells. Furthermore, we show that Swi1 is required for efficient chromatin association of Swi3 and that the Swi1 C-terminal domain directly interacts with Swi3. These results indicate that Swi1 associates with chromatin through its DDT domain and recruits Swi3 to function together as the replication fork protection complex.     

Cancer and the bromodomains of BAF180

Chromatin remodelling complexes alter the structure of chromatin and have central roles in all DNA-templated activities, including regulation of gene expression and DNA repair. Mutations in subunits of the PBAF (polybromo/Brg1-associated factor) or SWI/SNF-B remodelling complex, including BAF180, are frequently associated with cancer. There are six potential acetyl-lysine-binding BDs (bromodomains) in BAF180, which may function to target the PBAF complex to promoters or sites of DNA repair. In the present review, we discuss what is currently known about the BDs of BAF180 and their potential significance in cancer.     

Solution NMR structure of the barrier-to-autointegration factor-Emerin complex

The barrier-to-autointegration factor BAF binds to the LEM domain (Em(LEM)) of the nuclear envelope protein emerin and plays an essential role in the nuclear architecture of metazoan cells. In addition, the BAF(2) dimer bridges and compacts double-stranded DNA nonspecifically via two symmetry-related DNA binding sites. In this article we present biophysical and structural studies on a complex of BAF(2) and Em(LEM). Light scattering, analytical ultracentrifugation, and NMR indicate a stoichiometry of one molecule of Em(LEM) bound per BAF(2) dimer. The equilibrium dissociation constant (K(d)) for the interaction of the BAF(2) dimer and Em(LEM), determined by isothermal titration calorimetry, is 0.59 +/- 0.03 microm. Z-exchange spectroscopy between corresponding cross-peaks of the magnetically non-equivalent subunits of the BAF(2) dimer in the complex yields a dissociation rate constant of 78 +/- 2s(-1). The solution NMR structure of the BAF(2)-Em(LEM) complex reveals that the LEM and DNA binding sites on BAF(2) are non-overlapping and that both subunits of the BAF(2) dimer contribute approximately equally to the Em(LEM) binding site. The relevance of the implications of the structural and biophysical data on the complex in the context of the interaction between the BAF(2) dimer and Em(LEM) at the nuclear envelope is discussed.     

1H, 15N, and 13C resonance assignments and secondary structure of the SWIRM domain of human BAF155, a chromatin remodeling complex component

Mammalian SWI/SNF complexes are evolutionary conserved, ATP-dependent chromatin remodeling units. BAF155 in the SWI/SNF complex contains several highly conserved domains, including SANT, SWIRM, and leucine zipper domains. The biological roles of the SWIRM domain remain unclear; however, both structural and biochemical analyses of this domain have suggested that it could mediate protein-protein or protein-DNA interactions during the chromatin remodeling process. The human BAF155 SWIRM domain was cloned into the Escherichia coli expression vector pMAL-c2X and purified using affinity chromatography for structural analysis. We report the backbone ¹H, ¹⁵N, and ¹³C resonance assignments and secondary structure of this domain using nuclear magnetic resonance (NMR) spectroscopy and the TALOS+ program. The secondary structure consists of five α-helices that form a typical histone fold for DNA interactions. Our data suggest that the BAF155 SWIRM domain interacts with nucleosome DNA (K _d = 0.47 μM).